The invention provides a preparation process of a rabies vaccine without antibiotic addition in the whole process from a cytotoxic strain source. A Vero cell pre-master cell bank, a master cell bank,a working cell bank, a CTN-1V strain pre-master seed batch, a master seed batch and a working seed batch are prepared by using a culture medium without antibiotic addition, a bioreactor and a flaky carrier are applied, and through performing pre-culture and perfusion culture on Vero cells, inoculating a working seed batch of rabies viruses, performing washing and exchanging, performing perfusion culture to obtain a virus solution, performing ultrafiltration concentration, performing inactivating, performing purifying, preparing a semi-finished product, performing subpackaging and performing freeze-drying, the rabies virus vaccine is obtained. By optimizing the process of the virus adding course of bioreactor culture, impurity residues in the rabies vaccine are effectively reduced, the residual quantity of bovine serum components, Vero cell DNA and Vero cell host proteins is reduced, compared with other vaccine products on the market, the prepared vaccine product has superiority in clinical trial safety, and the safety of biological products is ensured to the greatest extent.