The rejection came less than a week after the Disney board said it would carefully consider the Comcast offer, which would have created the world's largest media company.
The board in a statement expressed support for current chairman and chief executive Michael Eisner and said the company's current structure and strategy are sufficient to maximize shareholder value. But it left open the possibility it would sell for a higher price.
"We are committed to creating shareholder value now and in the future and will carefully consider any legitimate proposal that would accomplish that objective," the statement read.
"In any proposal by Comcast, or any other company, the board will consider and assess the value to be received in exchange for the shares of Disney, and also the appropriate premium to reflect the full value of Disney."
The board noted that Comcast's offer to swap 0.78 of a share of Comcast for each Disney share undervalued the company by at least $6.6 billion based on the closing prices of both companies the day after Comcast announced its offer.
Disney's shares rose 15 percent last Wednesday — the day of Comcast's offer — then rose 1.4 percent on Thursday, and dropped 3.8 percent on Friday to close at $26.92 on the New York Stock Exchange.
Comcast's shares fell 12 percent from Wednesday through Friday to close at $29.90 on the Nasdaq Stock Market.
Comcast said in a statement Monday its offer "reflects a full and generous valuation based upon Disney's prospects and performance over a long period of time, representing a significant premium over Disney's unaffected share price during any relevant measurement period over the last three years.
"We maintain the belief that our merger proposal represents a sound and compelling proposition for both sets of shareholders," the statement said.
Comcast's bid was initially valued at $54 billion, with Disney shareholders retaining 42 percent ownership of the combined company. Comcast also said it would assume $11.9 billion in debt from Disney, which owns ABC, ESPN, movie studios and theme parks.
In its statement Monday, Disney's board of directors said that 0.78 of a share of Comcast is selling for $3.60 less than the market price of a share of Disney.
"The deficit of value in Comcast's proposal has existed from the very first day after Comcast announced it, when the deficit was $3.24 per Disney share or a total of $6.6 billion," the statement read.
Disney and Comcast together had $45 billion in revenues last year. If a deal had been reached to combine the companies, they would edge out Time Warner, which had $39.6 billion in revenues last year.