To express recombinant structural protein VP1 of enterovirus 71 (EV71) in E.coli and prepare anti-VP1 monoclonal antibodies (mAbs).With P1 gene as a template, EV71 VP1 gene was amplified by PCR and cloned into the expression vector pET-32a(+). After transformation into E.coli TG1, the positive clones were screened and sequenced. The recombinant plasmid was then transformed into BLgold (DE3) and the recombinant protein in inclusion bodies was induced by IPTG and detected by SDS-PAGE. After the inclusion bodies were solubilized with 6 mol/L guanidine hydrochloride, we purified VP1 protein using Ni-NTA affinity chromatography, and then immunized the mice with it to prepare the mAbs against VP1 protein.Recombinant VP1 protein was expressed in E.coli. We obtained totally 24 VP1 monoclonal hybridoma cell strains, of which one was EV71 positive determined by Western blotting and five were positive for IFA.These mAbs are valuable reagents for the development of new vaccines and detection kits for EV71.