Abstract: Objective To prepare and identify specific Phosphatase of Regenerating Liver-3 (PRL-3) monoclonal antibodies for clinical detection and for further therapeutic intervention. Methods Hybridoma technology was used to prepare PRL-3 monoclonal antibodies (MAbs)，and the specificities of MAbs against PRL-3 were evaluated by Western blot and immunoprecipation. Six truncations of PRL-3 were cloned and expressed in prokaryotic cell for identifying the approximate epitope. The binding abilities of MAbs were analyzed by Western blot. Results Among 9 hybridoma clones obtained, 6 （9D8, 9E2, 9F4, 11B2, 4D3 and 4D10）could specifically bind to PRL-3,and 4-could react to PRL-3 protein in eukaryotic cells.Clone 9D8,9E2,9F4 and 11B2 cloud bind to the COOH terminal of PRL-3，and clone 4D3 and 4D10 cloud bind to 69~95 amino acids. Conclusion MAbs 9D8, 9E2, 9F4, 11B2, 4D3 and 4D10 can react only to PRL-3, which provides potential applications in and will be powerful and reliable tools for future study and clinical diagnosis and in future study.