At present, there are no vaccines available for hand, foot, and mouth disease, which is caused by Coxsackie virus A16 (CVA16) infection. In the present study, we isolated epidemic strains of CVA16 and optimized the production of the virus in Vero cells. The system comprised growing the infected cells on polymer fiber paper carriers in a serum-free medium containing 0.5% (w/v) lactalbumin hydrolysate a mini bioreactor. Disposable Bioflo310 and AmProtein Current perfusion bioreactors were used to monitor virus infection and Vero cell culture. The total number of cells increased from 1.5 x 10(9) to 3.0 x 10(10). In our optimized culture process, the virus titer reached 7.8 x 10(7) TCID50/mL at three days after infection. The inactivated CVA16 prepared from our optimized culture procedure elicited a slightly higher neutralizing antibody titer compared with that derived from routine culture procedures. These results will promote the large-scale production of inactivated CVA16 vaccines using nonwoven polymer fiber paper cell cultures.