To prepare a monoclonal antibody against human SIRPα using synthetic peptide, and to use it for immune test to assess its efficacy. Methods The synthetic peptide of SIRPα was linked with KLH and the product was used as antigen for immunization of BALB/c mice. The mAb anti-SIRPα was obtained by hybridoma technique. The produced mAb was used for flow cytometry, Western blotting analysis and immunohistochemistry assay. Cytokines secreted by PMA-treated THP-1 cells were tested by antibody arrays after exposure to the obtained mAb, and the levels of TNF-α and IL-6 were assayed by ELISA. Results The mAb secreting hybridoma clone was successfully obtained and it had a satisfactory efficacy when used for flow cytometry, Western blotting analysis and immunohistochemistry assay. Compared with negative control and isotype control, the prepared mAb can stimulate TNF-α and IL-6 secretion in PMA-treated THP-1 cells. Conclusion We have successfully prepared the mAb against SIRPα using synthetic peptide.