Abstract: Objective To prepare Clostridium perfringens (CVCC60101) for production and testing of veterinary vaccines, verify its biological characteristics, and perfect the standard and method of preparation and verification. Methods In this study, Clostridium perfringens (C) prepared and stored by freeze-drying in 1960 was resuscitated, rejuvenated using susceptible animals, isolated, cultured, freeze-dried, and the new strain’s morphology, culture characteristics, biochemical properties, serological characteristics, virulence, immunogenicity, and other characteristics were tested. Results The result showed that the new strain was pure, and its morphology, culture characteristics, and biochemical properties accorded with the characteristics of Clostridium perfringens. In addition, its vacuum degree, residual moisture, serological characteristics, virulence, and immunogenicity accorded with the standard of the strain for production and testing of veterinary vaccines. Conclusions This research gives more experimental data for perfecting the standard and method of preparation and verification of Clostridium perfringens (C).