Renato Velasquez of Hanover Park did not speak as an attorney entered the plea Monday in a DuPage County courtroom.
The 46-year-old trucker faces multiple felony charges, including driving while fatigued and falsifying truck driving logs.
The Interstate 88 crash near Aurora killed maintenance worker Vincent Petrella and critically injured trooper Douglas Balder.
They were helping a disabled truck at the time when the crash happened.
Balder's wife attended Monday's hearing as did many of her husband's fellow troopers.
Illinois State Police Captain Patrick Kimes says Balder is improving but still in intensive care.
"Because of the extent of his injuries he is still being medicated to some degree, but he is slowly being brought out of that so that he is at some points during the day responsive to questions from the medical staff," Kimes said.
"My client plead not guilty today," Velasquez attorney Steven Goldman said. "I have to review the discovery there are approximately 600 pages of reports. There's more to come."
Velasquez is free on bond, but was ordered to surrender his passport Monday at the request of prosecutors.
He is due back in court April 14.