Joseph James DeAngelo, 72, appeared in an orange prison jumpsuit, handcuffed to a wheelchair.
He was formally arraigned on two murder charges dating back to 1978.
Mr DeAngelo is a suspect in 11 other murders and 51 rapes committed across California between 1976 and 1986.
Investigators linked him to DNA left at crime scenes, in part thanks to a genealogy database designed to trace family histories.
DNA sent to such a site by a relative of the murderer may have provided a crucial clue to police.
The Golden State Killer, also known as the East Area Rapist, Original Night Stalker, and the Diamond Knot Killer, terrorised communities in central, northern and southern California.
Mr DeAngelo spoke only briefly in court to confirm he had a lawyer. He did not enter a plea, and was denied bail.
Sacramento County Sheriff Scott Jones said Mr DeAngelo is on suicide watch in the psychiatric ward of the county jail. If convicted, he could face the death penalty.
Some survivors were present in court for the hearing, as well as victims' friends and family members.
One woman was pictured holding up a picture of two of the Golden State Killer's victims, Cheri Domingo and Greg Sanchez.
Mr DeAngelo's next court appearance is scheduled for 14 May. Authorities have said more charges are likely to be filed in due course.
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