The assembly of enterovirus requires the cleavage of P1 polyprotein by protease 3CD into individual structural proteins. Two recombinant baculoviruses were constructed to encode P1 and 3CD of enterovirus 71 (EV71), respectively. The expressed 3CD successfully cleaved P1 in vitro and in vivo. Also, the co-infection in insect cells resulted in crystalline virus-like particle structures morphologically resembling the authentic EV71 aggregates, which are reported for the first time.