During his short arraignment hearing, the 19-year-old suspect, clad in an orange jail jumpsuit, entered his plea to the 30-count federal indictment charging him with using weapons of mass destruction and killing four people.
Tsarnaev, his left hand bandaged, answered "not guilty" as each charge was read, NBC News reported.
He is to be back in court Sept. 23.
Liz Norden, whose sons, J.P. and Paul, each lost a leg in the dual explosions that killed three people and wounded 264 others near the marathon's finish line April 15, told WHDH, Boston, before the hearing she showed up in search of answers behind the terror attack.
"How can somebody do something to innocent people like that?" she said.
"I just feel I can maybe find some answer or a peace of mind by going."
Prosecutors have not decided whether to seek the death penalty for Tsarnaev, an American citizen born in Dagestan.
His brother Tamerlan, 26, was killed when run over allegedly by Tsarnaev as he escaped a shootout with police following the bombings.
Authorities allege the brothers were self-radicalized Muslims angered by the wars in Iraq and Afghanistan.
The former college student and his brother allegedly set the bombs that killed three people and injured scores more at the finish line of the Marathon on April 15. The brothers allegedly killed a police officer at the Massachusetts Institute of Technology as well.
Martin Richard, 8, Krystle Marie Campbell, 29, and Lingzi Lu, 23, a Chinese student, were killed in the bombing. Many of the injured suffered devastating wounds.
So many people came to the hearing with a line outside the courthouse by 7:30 a.m. that authorities set up an overflow room. Most of the spectators, including victims of the bombing and their families, were not sympathetic to Tsarnaev but some supported him.
Jennifer Mack of Boston wore a T-shirt with Tsarnaev's picture.
"This has all been set up. He's been framed," she said.