Objective To exclude false positivities in detection of IgM antibodies against hepatitis E vires of genotype 4 (HEV-4) using a truncated immunodominant polypeptide of HEV open reading frames (ORF2). Methods The recombinant ORF2 immunodominant polypeptide corresponding to amino acids (AA) 459-607 and a truncated polypeptide corresponding to AA 472-607 were separately applied to coat ELISA plates. Anti-HEV IgM from 35 serum samples with HEV RNA positive, 69 serum samples from healthy individuals and 117 clinically suspicious HEV RNA positive serum samples was detected by an indirect ELISA and was confirmed by western blot in protein level and RT-PCR detecting in RNA level. Results Western blot analysis showed that the sera from HEV patients reacted with the dimmer of peptide 459-607, but they didn't react with the monomer and peptide 472-607. The ELISA showed that all 35 serum HEV RNA positive samples reacted with peptide 459-607 but not with peptide 472-607 and none of the 69 serum samples from healthy individuals reacted with either polypeptide. Among 117 chnically suspicious HEV RNA serum samples, 5 samples reacted simultaneously with both polypeptides. But the difference between 450 nm absorbance (A450) value was less than 0. 5. Western blot analysis demonstrated that all the 5 serum samples were anti-HEV IgM- negative. The 5 serum samples was detected negative by RT-PCR, indicating that the false pesitivities were caused by non-specific absorption. Conclusions ORF2 peptide 459-607 may be used to detect anti-HEV lgM efficiently. The false positivities caused by non-specific absorption can be largely excluded according to the difference between 45Ohm absorbance (A450) value when serum reacts with both polypeptides.