PRL-3 is a newly identified nonclassical protein tyrosine phosphatase associated with colorectal cancer metastasis. To prepare specific monoclonal antibody (MAb) against PRL-3, cDNAs of PRL-1, -2, and -3 were cloned by RT-PCR and inserted into prokaryotic expression vector pGEX-4T1, respectively. The fusion proteins were expressed in Escherichia coli. Monoclonal antibody against PRL-3 was obtained with hybridoma technique and specific ELISA screening. Western blotting and immunohistochemistry assays showed that MAb 3B6 had specific binding ability with PRL-3 protein in eukaryotic cells. This MAb will be a helpful tool for the detection of PRL-3 protein in the tissues and serum of colorectal cancer and other cancer patients.