Enterovirus 71 (EV71) has led to recent outbreaks of hand, foot and mouth disease (HFMD) in China, resulting in high mortality. In this study, several monoclonal antibodies were generated by immunizing mice with two synthetic peptides, SP55 and SP70, containing amino acids 163-177 and 208-222 of VP1. The specificities of the anti-EV71 peptide monoclonal antibodies were confirmed by Western blot analysis and immunocytochemistry against EV71 virus. Most importantly, we have identified a monoclonal antibody, clone 22A12, which shows strong neutralizing activity against EV71 in an in vitro neutralization assay. Because there is no vaccine available and treatment is very limited, mouse anti-EV71 monoclonal antibody, clone 22A12, could be a promising candidate to be humanized and used for treatment of EV71 infection.