Schenecker, 50, appeared in court in Tampa, Fla., dressed in a red jail outfit, The Associated Press reported. The judge appointed the public defender's office to represent her.
If convicted, Schenecker could face the death penalty.
Police found the bodies of Schenecker's two children, Calyx, 16, and Beau, 13, in the family home on Jan. 28. Both children had been shot in the head. Police say Schenecker told investigators that she killed the children because she was tired of them talking back.
The not guilty plea means that the burden of proof remains with the prosecutors, the St. Petersburg Times reported.
Schenecker sat with her eyes closed during the hearing, according to The Tampa Tribune.
About a dozen deputies attended the hearing, with two keeping a firm arm on Schenecker's arms. The extra security was in place partly because of fears for Schenecker's safety, sheriff's spokesman Larry McKinnon said.
"It's because of the extreme emotions this case causes and the national attention it has received," McKinnon said.
Robert Frasier, an assistant public defender representing Schenecker, says he will apply for a motion to freeze the family's assets until it can be decided who will pay for her defense, the AP said.
Authorities say that Schenecker shot Beau as she was driving him back from soccer practice on the evening of Jan. 27. Police say he had been talking back to Schenecker during the trip.
When she got home, Schenecker allegedly went up to Calyx's bedroom, where the girl was studying. Authorities say Schenecker then shot Calyx in the head.
Julie Schenecker's husband, U.S. Army Col. Parker Schenecker, was not in the house at the time of the killings, as he was overseas. Parker Schenecker did not attend today's hearing, according to the St. Petersburg Times.
"He is meeting with family members and close friends to formalize his plans to honor the memory of Calyx and Beau," a family spokeswoman said. "At the same time, he is considering the best path to help him heal from this tragedy."

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