Objective To prepare a neutralizing monoclonal antibody (mAb) that can simultaneously block enterovirus 71 (EV71) and coxsackievirus A16 (CV-A16) infections. Methods BALB/c mice were immunized with 163-177 amino acids (SP55) of the C-terminal of EV71 virion particle 1 (VP1) protein, and the mAbs were prepared by hybridoma technology. Neutralization antigenic epitope SP55 of EV71 and the highly homologous CV-A16 VP1 protein C-terminal 163-177 amino acids (PEP55) were applied to detect the mAbs that cross-reacted with EV71 and CV-A16 at the same time, and an in vitro neutralization test was conducted to detect the neutralization effect of EV71 and CV-A16, and to analyze the biological characteristics of the mAb. Results A mAb 6E5 with IgG1 subclass heavy chain and Kappa light chain was prepared, 6E5 mAb can cross-neutralize both EV71 and CV-A16. The mAb 6E5 could neutralize EV71 with a titer of 1:128, and CV-A16 with a titer of 1:32. Conclusion We have prepared a mAb 6E5 with a pan-spectrum neutralizing activity that can neutralize EV71 and CV-A16 at the same time.