Fioramonti, of the anti-establishment Five Star Movement, wrote on Facebook that he was stepping down because his demand for a minimum level of funding had not been met.
He said he had informed Prime Minister Giuseppe Conte of his decision on Tuesday.
Fioramonti had agreed to take on the portfolio to “reverse… the trend that has for decades put Italian schools, higher education and research in conditions of great suffering,” he said.
He said the government had failed “to ensure a financial waterline… especially in such a crucial area as universities and research.”
The resignation is a new blow to the coalition government formed by Fioramonti's M5S party and the centre-left Democratic Party just four months ago.
Dissensions have already arisen in several areas including migration.
M5S leader Luigi Di Maio, who is foreign minister, has come under harsh criticism within the party, with several lawmakers leaving to join the far-right, anti-immigrant League party led by Matteo Salvini.
Media reports say Fioramonti plans to form an independent group in parliament to support Conte that could be the embryo of a new political party.
Fioramonti, a former economy professor at the University of Pretoria in South Africa, had set a minimum 2020 budget of three billion euros ($3.3 billion), but was accorded two billion in the budget approved on Monday.
“It seems resources can never be found when it comes to schools and research, and yet hundreds of millions of euros are available within a few hours for other objectives when there is the political will,” he said.
His resignation will likely raise tensions within Italy's fractured coalition government as it faces a challenge by former prime minister Matteo Renzi and his new centrist party, Italia Viva.
Meanwhile, Salvini is calling for new elections even though the League has declined in opinion polls since he broke up Italy's previous coalition government in August.