<p>Background: This study aimed to develop novel anti-coagulant protein based on phospholipid binding protein and thrombin inhibitor. Methods: Recombinant protein composed of hirudin and annexin A5 (r-HirA5) was obtained by genetic engineering and SEC-HPLC profile was used to determine the aggregation morphology. The retention of antigenic determinants was identified by Western blot analysis. The binding specificity analysis of different kinds of phospholipid molecules, phospholipid binding dose analysis, and the effect assay of Ca2+ concentration were performed to identify the characteristics of phospholipid affinity. The plasma coagulation time detection, blood clotting assay, and anti-thrombin activity determination were performed. Results: r-HirA5 was structurally stable and existed in solution as a monomer, preserving the distribution of antigenic determinants of the annexin. r-HirA5 retained most phospholipid binding properties and showed limited in vitro thrombin binding activity. r-HirA5 demonstrated good anti-coagulant effects such as delayed plasma coagulation time and prolonged APTT value. Conclusion: The recombinant protein r-HirA5 exhibits stable physiochemical characteristics and good anti-coagulant activity. This study provides the clue for the development of novel targeted anti-coagulants for thrombotic disease.</p>