Objective To prepare the monoclonal antibodies specifically against P protein of human respiratory syncytial virus (HRSV) and identify it. Methods HRSV P protein prepared by prokaryotic expression in the form of overlapping peptides was used as the immunogen, and monoclonal antibodies (mAbs) were screened by hybridoma technology. Western blot analysis was used to verify the binding activity of the screened mAbs and P protein, and immunofluorescence cytochemical staining was used to determine whether the obtained mAbs could be used to detect the expression of P protein in HEp-2 cells infected with HRSV. Results P181-15A3 and P211-16D8 with great reactivity and specific recognition of HRSV P protein were screened. Both mAbs could bind to P protein by Western blot analysis and could be used for immunocytochemical detection of P protein in HEp-2 cells after HRSV infection. Conclusion We have successfully prepared the mAbs against HRSV P protein.