The Chihuahua state Attorney General's Office said the ambulance driver was shot in the head and appeared to be the target of the attack. It did not provide theories about a motive.
Two of the victims were patients being taken to a Ciudad Juarez facility for kidney treatment, officials said. Also killed was a woman accompanying the patients.
Rosendo Gaytan, a spokesman for the Mexican Social Security Institute in Ciudad Juarez said a pickup truck carrying the gunmen intentionally crashed into the ambulance, forcing it to stop. The attackers then got out of the truck and opened fire on the ambulance, Gaytan said.
Ciudad Juarez, which is across the border from El Paso, Texas, is the site of a war between the Juarez and Sinaloa drug cartels and saw some 3,100 homicides last year. Killings have gradually decreased but violence remains high.
Authorities reported six other slayings Wednesday in addition to the ambulance attack.
Elsewhere, three members of a criminal gang allied with the Zetas drug cartel have been detained in connection with the slayings of 26 people last month in the western Mexican city of Guadalajara, prosecutors said Wednesday.
Jalisco state Attorney General Tomas Coronado said the arrested men belonged to the Milenio gang and had told police that the slain men were members of a rival group that refused to join with Milenio.
Analysts have described the Guadalajara slayings as the result of a growing rivalry between the Zetas and the powerful Sinaloa cartel. The Sinaloa cartel is based in Sinaloa state, to the north of Jalisco.