A statement released by the Pakistan Foreign Office said Dr Afridi had caused immense damage to the country's anti- polio drive.
The fresh skirmish comes days ahead of the Ministerial level Strategic dialogue between the two countries beginning on January 27 in Washington after a delay of over three years.
The Consolidated Appropriations Bill 2014, approved by US Congress, was signed into law by US President Barack Obama on January 17.
The State Department will now undertake the process of making allocations, including those for Pakistan.
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"However, we are disappointed that the bill proposes to withhold USD 33 million from assistance on account of Dr Shakil Afridi's detention.
"Afridi, a citizen of Pakistan, is accused of having violated the country's laws. His action also caused immense damage to the polio campaign in the country. His case is sub judice and he remains entitled to due process under the law. Consequently, any linkage of US assistance to this case is not in keeping with the spirit of cooperation between the two countries," the statement said.
The doctor, who was arrested immediately after the May 2, 2011 operation by US commandos that killed then al-Qaeda leader bin Laden, has been convicted for treason over alleged ties to militant group Lashkar-e-Islam.
A tribal court in the semi-autonomous Khyber Agency had sentenced Afridi, who is currently being held at the central prison in Peshawar.
Legal experts and rights activists had challenged the verdict. The US has been pressing Pakistan for the release of Afridi.