Objective:To study the solid-phase synthesis of the thrombin inhibitor bivalirudin using Wang Resin and Fmoc-protection strategy. Methods:Bivalirudin was synthesized through the process of coupling reaction,TFA cleavage/deprotection and HPLC purification. Different Fmoc-Leu substitution resins (0.29 mmol/g,0.48 mmol/g,0.59 mmol/g) and different coupling reagents (HBTU,DCC,HATU) were used to synthesize the bivalirudin. Results:The purity of bivalirudin could be >98%. The bioactive assay showed that the synthetic bivalirudin had the same extent anticoagulant activity as the standard sample. The results showed:① the high Fmoc-Leu substitution resin (>0.5 mmol/g) results in the low purity product,which was also difficult to purify;the substitution 0.3-0.4 mmol/g of Fmoc-Leu-Wang Resin was recommended;②using HBTU/HOBt as the coupling reagent had the advantages of convenient operation,economic and high efficiency. Following the results above,the optimized condition was used to synthesize gram grade of the Bivalirudin using 5 g Fmoc-Leu-Wang Resin. The crude product with the 84% total yield and 68% purity was obtained. Conclusion:The solid-phase synthesis method was used to evaluate the effect of different Fmoc-Leu substitution resins,different coupling reagents and different synthesis size on the total yield,purity and activity of bivalirudin. This research has instruction for the synthesis of bivalirudin in large amount.