Ullah, 27, faces a litany of charges in the December 11 bombing in an underground walkway connecting two subway lines beneath the Port Authority Bus terminal.
When Manhattan Federal Judge Richard Sullivan asked Ullah his plea, he replied, "At this moment, not guilty."
This was his first appearance before a New York court. Days after his arrest he had appeared through video conference. Ullah faces life in prison if convicted of the attack that injured five persons.
He was also injured in the explosion, and has been in federal custody since his arrest.
On Wednesday, a federal grand jury indicted Ullah with one count of providing and attempting to provide material support to the Islamic State and one count of using and attempting to use a weapon of mass destruction.
He has also been charged with one count each of bombing and attempting to bomb a place of public use and a public transportation system; destruction and attempted destruction of property by means of fire or explosives; conducting and attempting to conduct a terrorist attack against a mass transportation system.
"As alleged in the indictment Akayed Ullah constructed a pipe bomb and detonated it in a mass transit hub in the heart of New York City to terrorise as many people as possible and to bring ISIS-inspired violence to American soil," said Acting Assistant Attorney General Dana Boente.
Authorities said the explosion was an isolated attempted terrorist attack. Ullah posted on Facebook earlier in the day of the attack, "Trump you failed to protect your nation," according to a criminal complaint.
He is expected back in court on April 13 for a status conference.
He began to self-radicalise three years ago, authorities said, but law enforcement said he was not on their radar before the subway attack.
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