To determine the B cell epitope of a monoclonal antibody against the M3-M4 loop of NMDAR1, a random phage displayed dodecapeptide library was screened with the monoclonal antibody MAB363 against the M3-M4 loop of NMDAR1. After four rounds of biopanning，the peptide sequences of positive phage clones were determined and analyzed by DNA sequencing，ELISA and competitive inhibition assay. A positive clone was found (clone1: VHTNPSTWQPIL). The binding between clone1 and MAB363 were competitively inhibited by the recombined M3-M4 loop expressed by E.coli DH5α；The binding between M3M4 and MAB363 could be competitively inhibited by one of solid-synthesized epitope peptides: RLRNPSKD. There were identical sequences among them: NPS. Deleted with NPS, the peptide could not inhibit the binding of MAB363 to M3-M4. These results demonstrate that NPS in M3-M4 loop is the B cell epitope recognized by MAB363，which may be important for developing a practical immunization strategy against excitotoxic brain injury.