Programmed death-1 (PD-1) is a transmembrane protein that shares homology with the B7/CD28 family of T cell signaling molecules. PD-1 interacts with its ligands PD-L1 and/or PD-L2 and provides a negative regulatory signal to CD4 and CD8 T cells that results ultimately in a phenotype termed T cell exhaustion. Here we expressed and purified mouse PD-1 protein and developed a monoclonal antibody (MAb) against mouse PD-1 by immunizing BALB/c mice with a specific region of the extracellular domains of PD-1 as antigen, which was expressed in Escherichia coli. A stable hybridoma cell line was established by animal immunization, cell fusion, and hybridoma screening. The MAb was then prepared from mouse ascites after inoculating the hybridoma cells. Different methods were used to analyze the characterization of the MAb, including ELISA, Western blotting, flow cytometry, and RT-PCR techniques. The results showed that the PD-1 MAb can bind to the PD-1 protein and promote lymphocyte proliferation. This PD-1 MAb will be a valuable tool for further investigation of programmed death-1 functions.