Tsarnaev, 19, entered the plea Wednesday in federal court in Boston.
For the first charge, he leaned toward a microphone and said, "Not guilty," in a Russian accent. He then said not guilty repeatedly about a half-dozen more times.
Federal prosecutors are weighing whether to pursue the death penalty.
Police believe he and his brother, Tamerlan, carried out the April bombings, which killed three people and injured more than 260.
Tamerlan was killed days after the bombings during a shootout with police.
The courthouse was jammed for Tsarnaev's appearance. A spokeswoman for the U.S. attorney's office said space was reserved in the main courtroom for victims' families. Court officials also set aside an overflow courtroom to broadcast the hearing for the media.
Reporters and spectators started lining up for seats in the courtroom early in the morning, as Federal Protective Service officers and bomb-sniffing dogs surrounded the courthouse.
This was Tsarnaev's first public appearance since he was arrested April 19 after he was found bleeding and hiding in a boat in a suburb days after the April 15 explosion.
His initial court appearance took place at a hospital, where he was recovering from injuries suffered in a shootout with police the day before in Watertown.
The three killed were Martin Richard, 8; Krystle Marie Campbell, 29; and Lingzi Lu, 23. Authorities say the Tsarnaevs also killed Massachusetts Institute of Technology officer Sean Collier days later while they were on the run.
Numerous bombing victims had legs amputated after the two explosions, which detonated along the final stretch of the race a couple hours after the elite runners had finished.
The Associated Press contributed to this report.