Duck hepatitis A virus (DHAV) infection is characterized by an acute, rapidly spreading that affects young ducklings. DHAV-1 or DHAV-3 infection is prevalent, and simultaneous co-infection with both viruses has recently become increasingly frequent in the domestic duck farms. In this study, we developed a bivalent live attenuated vaccine (DHV-HSBP100 and AP-04203P100) for DHAV-1 and DHAV-3 and reported the protective efficacy and safety of the vaccine. At 1-day-old, the ducklings received a bivalent vaccine via intramuscular injection. The immunized ducklings showed effective and rapid protection against virulent DHAV-1 and DHAV-3 at 2 or 3 days post vaccination. Moreover, the ducklings showed a potent humoral immune response that peaked at 3 weeks and were maintained at 6 weeks after vaccination. The bivalent vaccine was safe; ducklings administered 10 doses of bivalent vaccines showed no clinical signs, mortality, gross lesions, and body weight changes compared with those observed in the negative controls. Ducklings vaccinated with a bivalent vaccine were evaluated for tissue tropism and viral replication of vaccine strains. Both bivalent vaccine strains were detected in various organs, and the highest virus replication was detected in the kidneys, among the tested organs. No interference occurred during the replication of both vaccine strains. Thus, these experiments suggest that bivalent vaccines would be useful as a promising and practical strategy for control DHAV outbreaks caused by DHAV-1 and DHAV-3 in duck farms.