The neuraminidase (NA) of influenza virus is a receptor-destroying enzyme, removing sialic acid from carbohydrate chains attached to HA, and releasing the viruses from infected cells. The NA genes of the 2009 A H1N1 were sequenced, then the B-cell epitopes were predicted, screened and assessed based on immunoinformatics. Two peptides SE8 and RE6 (amino acid residues 168～175 and 428～433) of NA protein were synthesized and immunized to raise antisera in rabbits. Two antisera are capable of eliciting neutralizing antibodies against 2009 A H1N1 in the in vitro microneutralization assay, furthermore, the anti-releasing effects of hemagglutination existed in the antisera. Alignment with databases showed that the amino acid residues of two epitope peptides are highly conserved amongst the NA sequences of the strains isolated from the world. These findings indicate that SE8 and RE6 represents an attractive candidate for an effective synthetic peptide-based vaccine against 2009 A H1N1 viruses.