Thrombin (THR) inhibitors play an important role in the treatment of thrombotic diseases. This study established a THR-based bio-specific extraction coupled with affinity chromatography and ultra-high performance liquid chromatography-high resolution mass spectroscopy (UPLC-HR-MS) analysis method to screen and identify THR ligands in Leech. After evaluating the reliability of the screening method using positive control drug (hirudin), it was successfully used to screen the potential active constituents in leech. And a comprehensive analysis of the peptides in leech elution was performed by UPLC-HR-MS, a total of 34 peptides were identified. At the same time, anti-THR activity was explored and inferred by searching databases and published literature. As a result, six peptides were discovered to be potential active compounds in leech. Further, the six peptides were synthesized and in vitro enzymatic activity assay was performed. Finally, SYELPDGQVITIGNER was screened as an anti-THR peptide with an IC50, value of 255.75 mu M and it was discovered for the first time from Whitmania pigra Whitman and Hirudo nipponica Whitman. The molecular docking study showed that THR inhibitory activity of the polypeptide was mainly attributed to the hydrogen bond interactions, van der Waals forces and electrostatic interactions interaction between polypeptide and THR. These results suggest that the polypeptide is a potential natural THR inhibitor that can be used as anticoagulant.