The denial came with former President Carter due in Cuba Sunday.
Speaking live on state television, Castro called on U.S. officials to "present even the most minimum proof" of the allegation Undersecretary of State John Bolton made Monday.
"The only thing true in Bolton's lies is that Cuba is 90 miles away from United States territory," said Castro, adding that the United States would be unable to provide evidence of the claim because such evidence "does not and cannot exist."
"No one has ever presented a single shred of evidence that our homeland has conceived a program that develops nuclear, chemical or biological weapons," Castro said. "The doors of our institutions are open ... Cuba has absolutely nothing to hide."
Castro's speech Friday was Cuba's first detailed response to the charges. In a brief note on Thursday, Havana had simply described Bolton's statements as "loathsome."
The Cuban government called out more than 100,000 people for a Saturday morning rally in a Havana suburb to denounce Washington's "fallacies."
At the rally, the directors of Cuba's top research laboratories denied the charges as well, saying they're dedicated to developing medicines to save lives - not killing people.
"To say that we in Cuba are making biological weapons ... is a simple and complete lie," declared Dr. Agustin Lage, director of Cuba's Molecular Immunology Institute. He described the U.S. charges as "very grave" and said many American scientists had visited Cuba's research institutes in the past and knew the charges were false.
A half-dozen other top scientists issued similar denials at the rally in the community of Regla, located across the bay from the capital of Havana.
In his Monday address to the Heritage Foundation, a conservative research group in Washington, Bolton said the Bush administration believes Cuba is trying to develop biological weapons and transferring its technical expertise to countries hostile to the United States.
The accusation marked the first time the U.S. raised the possibility of involvement by Cuba, the only outright U.S. foe in the Western Hemisphere, in attempts to develop weapons of mass destruction.
It seemed aimed at adding to the Bush administration's rationale for keeping Cuba on a list of countries accused of engaging in international terrorism.
Castro said his country opposes terrorism and noted that Cuba publicly condemned the Sept. 11 terrorist attacks the same day they took place.
He said any Cuban scientist discovered to be working on biological weapons for the transfer to other countries would be tried for treason.
The Cuban leader described the U.S. as "a superpower that has thousands of nuclear weapons ... but cannot vanquish the human being."
Castro's government in the past has accused the U.S. of using biological means to destroy crops and livestock on the island.
Castro's comments came on the same day that Cuban activists mounted an unprecedented challenge to his 43-year-old rule, delivering a petition to the legislature demanding a referendum for broad changes in Cuba's one-party socialist system. The effort is known as Project Varela.
The referendum would ask voters if they favor civil liberties such as freedom of speech and assembly, and amnesty for political prisoners.
The petition, with more than 11,000 signatures, was handed in two days before a planned visit from former President Carter.
For more than two decades, he has worked to improve U.S. relations with communist Cuba. He'll will give it another shot when he arrives Sunday at the invitation of Castro.
During his 1977-81 presidency, Mr. Carter helped re-establish diplomatic missions in both countries, negotiated the release of thousands of political prisoners and made it possible for Cuban exiles to visit their relatives on the island and, for a short time, other Americans to travel here freely.
But a U.S. trade embargo is still in place after four decades and relations are as chilly as they've ever been. So the 77-year-old Mr. Carter seems determined to try to plant the seeds for future dialogue when he meets with Castro, 75.
Mr. Carter will be the first U.S. president - in or out of office - to visit Cuba since the 1959 revolution that put Castro in power. Calvin Coolidge was the last American head of state to come, in 1928.
During his visit, Mr. Carter "will have the opportunity to contact and meet with as many citizens as he wants to," the Cuban Communist Party's newspaper said.
Wayne Smith, who was the chief U.S. diplomat to Havana during the Mr. Carter administration, said he didn't expect "any miracles." But, he added, "Mr. Carter cannot achieve less than (President) Bush has, which has been zero."
Although Mr. Carter has emphasized this is a private visit and he will not be negotiating with the Cuban government, people on all sides of the debate are pressuring him to push their agendas.
The Bush administration, backed by Cuban exiles in the U.S., has hardened the U.S. stance toward Havana, promising not to ease trade sanctions until Cuba holds free elections and releases political prisoners.
The White House and the exiles want him to talk bluntly with his host about human rights and democracy - two of Mr. Carter's favorite subjects.
Exile groups also hope Mr. Carter will bring up Project Varela.
And Cuban officials and a growing number of Americans who oppose U.S. sanctions hope Mr. Carter will publicly condemn the trade embargo.
Politicians in the U.S. are debating whether to ease it, with powerful business lobbies seeking an end to restrictions on credit and travel by Americans to the island.