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.
He appeared in shackles and an orange prison suit, and replied "not guilty" as the charges were read to the court, BBC reported.
Prosecutors could press for the death penalty for seventeen 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, the report added.
People appeared outside the federal courthouse in Boston since early morning to claim a seat inside the court and two overflow rooms for a hearing that lasted just seven minutes, the report said.
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, it added.
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.
The brothers are from a family of ethnic Chechen Muslims from Russia and had been living in the United States for about a decade.
More than 260 people were injured when two pressure cooker bombs packed with nails, ball bearings and other shrapnel were detonated at the finish line of the Boston Marathon, the biggest terror attack in terms of mass casualty on US soil since 9/11 strikes, report added.
Image: BostonMarathon bombing survivor Karen Brassard arrives at the federal courthouse for the court appearance by accused Dzhokhar on Wednesday
Photograph: Brian Snyder/Reuters
Boston bombings suspect indicted on 30 counts
Renewed US demand to lift Infosys, IT sector outlook
Why India needs to speak up!
IN PHOTOS: Fireworks, parades and protests mark US I-Day
In PHOTOS: Obama surprises young chefs at 'state dinner'