A US official said the move against the US Agency for International Development (USAID) "harms the Bolivian people".
In a May Day address, Mr Morales accused USAID of seeking to "conspire against" Bolivia.
He linked the expulsion to a recent comment by the US secretary of state.
But US state department spokesman Patrick Ventrell defended USAID's work in Bolivia.
"We think the programmes have been positive for the Bolivian people, and fully co-ordinated with the Bolivian government and appropriate agencies under their own national development plan," he said.
USAID said it deeply regretted Mr Morales' decision.
"Those who will be most hurt by the Bolivian government's decision are the Bolivian citizens who have benefited from our collaborative work on education, agriculture, health, alternative development, and the environment," it said in a statement.'US backyard'
USAID has been working in Bolivia for almost five decades, and had a budget of $52.1m (£33.4m) for the country in 2010, according to its website.
It cites as its main aims the strengthening of Bolivia's health system and the provision of "equal access to health care by eliminating social exclusion", as well as improving "the livelihoods of economically and socially disadvantaged people by increasing income and managing natural resources".
On previous May Days, Mr Morales had announced the nationalisation of key industries, such as hydroelectric power and the electricity grid.
Speaking at a rally in La Paz on Wednesday, Mr Morales said he "would only nationalise the dignity of the Bolivian people".
He said there was "no lack of US institutions which continue to conspire against our people and especially the national government, which is why we're going to take the opportunity to announce on this May Day that we've decided to expel USAID".
He then turned to his Foreign Minister, David Choquehuanca, and asked him to inform the US embassy of his decision.
The president also linked the expulsion to a recent remark by US Secretary of State John Kerry, who referred to Latin America as "the backyard" of the US.
Mr Kerry made the remark as he tried to persuade US Congressmen of the importance of the region, telling them that "the Western Hemisphere is our backyard. It's critical to us."
Bolivia's ABI state news agency reported that USAID was "accused of alleged political interference in peasant unions and other social organisations".
Mr Morales has threatened USAID with expulsion in the past, saying that its programmes have "political rather than social" ends. He has also accused it of "manipulating" and "using" union leaders.
Mr Morales, who heads his country's union of coca growers, has been critical in the past of US counter-narcotic programmes in Bolivia, repeatedly stating that the fight against drugs is driven by geopolitical interests.
In 2008, Mr Morales expelled the US ambassador and Drug Enforcement Administration (DEA) for allegedly conspiring against his government.
Mr Morales became Bolivia's first indigenous president in 2005.
He was re-elected by a landslide in 2009, but has since faced protests from indigenous communities angered by the construction of a major road through their territory, and by police and army officers demanding better pay.
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