Objective: To develop a continuous flow sucrose d. gradient centrifugation purification process for freeze-dried human rabies vaccine (chicken embryo fibroblasts). Methods: The effects of concentration of initial sucrose solution and loading speeds on the purification of Rabies virus (RABV) were compared to determine the purification process. The sample collection range was determined by multi-batch experiments The impurity removal and antigen recovery were compared under different concentration multiples to determine the appropriate concentration ratio of the harvest liquid The impurity removal rate and repeatability of the different batch samples after purification were compared to determine the stability of the purification process. Results: When the initial sucrose concentration is 60%, the 10-fold concentrated virus could be effectively purified at 150-200 mL/min. The removal rates of ovalbumin, bovine serum albumin and gentamicin reached resp. 99%, 95% and 95%. Conclusions: The continuous flow sucrose d. gradient centrifugation technique developed in this study can be used as an industrial purification process for freeze-dried human rabies vaccine (chicken embryo fibroblasts).