Gordon Duguid, a US state department spokesman in Washington, said Morales' accusations were "baseless" and that the embassy had not received any request for Philip Goldberg to leave.
Morales said he had asked his foreign minister to send a letter to the US embassy asking Goldberg to "urgently return to his country".
The Bolivian leader did not offer specific evidence against Goldberg, but he has long accused him of conspiring with the country's conservative opposition.
In June, the government terminated US aid programmes in the coca-growing Chapare region aimed at encouraging farmers not to grow the crop for cocaine production.
Farmers had condemned the programme as ineffective.
Last week, Goldberg met Ruben Costas, the governor of Santa Cruz, Bolivia's richest province and the centre of a pro-autonomy revolt against the government.
South America's poorest nation has been in the grip of political turmoil for months with rebel state governors calling for a bigger share of energy revenues for their regions.
Last month, Morales convincingly won a referendum on his rule but in the rebel states, voters also returned most of the governors forming the opposition coalition.
After failed negotiations to find a compromise solution, Morales announced a new referendum, to take place in December, to vote on his rewritten constitution, which would redistribute land and national revenues to give more to the indigenous population.