Tsarnaev, 19, faces 30 counts of using a weapon of mass destruction in the two 15 April blasts that killed three, including an eight-year-old boy, reports the BBC.
He appeared in shackles and an orange prison suit, and replied "not guilty" as the charges were read to the court.
Prosecutors could press for the death penalty for 17 counts.
The suspect has also been charged over the death of a fourth person, a university police officer, who was allegedly shot dead by Tsarnaev and his brother Tamerlan in the days after the attack.
He is also charged in a carjacking incident and with downloading internet material from Islamist radicals some time before the blasts.
People appeared outside the federal courthouse in Boston as early as 07:30 EST (12:30 BST) to claim a seat inside the court and two overflow rooms for a hearing that lasted just seven minutes.
Tsarnaev arrived at court with his face swollen and his arm in a cast.
Two of the suspect's sisters watched the proceedings. One sobbed during the hearing while the other held a baby.
Before he was led out of the courtroom, the suspect seemed to smile and to gesture a kiss to his family members in the room.
Among the crowd was a young friend of Tsarnaev, Hank Alvarez, 19. He said: "Just knowing him, it's hard for me to face the fact that he did it."
Tsarnaev, a US citizen, was not in court last month during an indictment hearing, when a federal grand jury agreed that he should be tried on 30 charges.
His first court appearance took place at his hospital bedside, where he was recovering from injuries suffered in a shootout with police during the manhunt. He was later transferred to a prison hospital near Boston.
Tsarnaev's older brother Tamerlan, 26, was killed days after the attack during a massive police operation. He is also suspected of carrying out the attacks.
Authorities say the accused ran over his older brother as he fled the shootout in a hijacked car.
Dzhokhar Tsarnaev was found the next day, 19 April, hiding in a boat in a residential garden in Watertown, Massachusetts.
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