Canine distemper virus (CDV) causes a highly contagious and fatal disease in domestic and wild carnivores. Currently, vaccination is the primary method for preventing canine distemper. However, incidents of vaccine immunization failures continue to be reported. There are no specific and effective treatment agents available for canine distemper infection. Neutralizing antibodies offer a potential approach for the treatment of viral diseases. In this study, single B cell antibody technology was applied to obtain whole-canine antibodies against CDV. 7 monoclonal antibodies were screened and showed high binding affinity to CDV hemagglutinin (H) protein, with D16 and F53 exhibited high specificity and neutralizing activity against CDV. Furthermore, D16 exhibited effective therapeutic potential in dogs subjected to lethal dose CDV attacks in vivo. In conclusion, our study offers an alternative approach for acquiring neutralizing antibody and provides a promising new strategy for the treatment of CDV infection.