Spinal cord injury and regeneration related protein No. 69 (SCIRR69) is a rat transmembrane bZIP transcription factor homologous to mice and human transcription factor CREB3L2. Previous work demonstrated the N-terminal region plays a critical role in its transcriptional activity. In this study, a peptide containing 18 amino acids (5-22aa) at the N-terminus of rat SCIRR69 was synthesized and coupled to the carrier protein as immunogen. One hybridoma cell line was obtained by standard cell fusion technique, followed by enzyme-linked immunosorbent assay (ELISA) and Western blot screening. The newly developed monoclonal antibody (MAb) was designated 4B4, the isotype of which was IgG2a. Immunofluorescence and Western blotting results showed that MAb 4B4 could recognize the SCIRR69 protein in both native and denatured forms. 4B4 will be a useful tool for the functional research of SCIRR69 in future studies.