Abstract: An optimized baculovirus/insect cell-mediated system is provided for the production of enterovirus 71 virus-like particles to produce a vaccine against recent EV71 virus outbreaks. Co-expression of the viral capsid polyprotein P1 ORF derived from a fatal case in the Fuyang province of the People's Republic of China plus the 3 CD protease of EV71 prototype strain BrCr resulted in the formation of VLPs. The yields were increased by co-expression of both P1 and 3CD in separate transgene cassettes arranged in opposite orientation in a bicistronic baculovirus vector and by inserting the translational enhancing signal L21 in front of the capsid protein open reading frame. Faster transgene processing was achieved by using insect Sf21 cells instead of Sf9 cells.