The younger sibling witnessed the shooting in Homestead, Florida, according to the Miami-Dade Police Department.
The yellow school bus didn't have surveillance cameras, police said.
The 13-year-old girl was airlifted to Miami Children's Hospital, where she died, police said.
A motive hadn't been determined Tuesday, and police were investigating the relationship between the shooter and the victim, Officer Alvaro Zabaleta told reporters.
"It's traumatic for them for what they had to witness to begin with and now you're going to have to question them to ask them exactly what they saw," Zabaleta told CNN affiliate WPLG. "You know, it's tragic. Anytime it deals with a child, and then, of course, when we get the phone call that they confirmed that she passed away, it's very sad."
The victim was being bused about 6:30 a.m. Tuesday to Palm Glades Preparatory Academy Charter School in Miami, said Javier Baez, spokesman for Miami-Dade Police.
None of the other children on the bus was injured, and all parties present at the shooting were taken to the homicide bureau for questioning, police said.
Police didn't have further information Tuesday on the firearm used in the shooting.
Police say they have the suspected shooter in custody.