The aim of this study was to prepare and identify a monoclonal antibody against the extracellular domain of p75 neurotrophin receptor (p75NTR-ECD), which will be used in diagnostics, therapeutics, and as a tool in understanding the role of P75NTR in pathogenesis of neuronal degenerative diseases and cancers. In this study, hybridoma technique was used for production of anti-p75NTR-ECD monoclonal antibody. BALB/c mice were immunized with p75NTR-ECD recombinant protein. Hybridoma clones were screened using indirect enzyme-linked immunosorbent assay (ELISA). Anti-p75NTR-ECD monoclonal antibody was produced by ascites revulsion. Protein A affinity chromatography was used for the purification of anti-p75NTR-ECD monoclonal antibody. Titer of anti-p75NTR-ECD was assessed by ELISA. Specificity of anti-p75NTR-ECD was detected by immunofluorescence and immunohistochemistry. As a result, one stable hybridoma cell clone (3B5F9) producing anti-p75NTR-ECD monoclonal antibody was established. The titer of anti-p75NTR-ECD monoclonal antibody is 1:51200. A 2.91 mg monoclonal antibody against p75NTR-ECD with high specificity was prepared. Immunofluorescence and immunohistochemistry showed that p75NTR-ECD positive staining occurs in the plasma membrane of glioma cell and tissue, which results in an advantage in diagnostic and therapeutic targeting of P75NTR expressing neuronal degenerative diseases and cancers.