Background: To observe the effect of highly ag-glutinative staphylococcin (HAS) combined with α-calcium sulfate hemihydrate (α-CSH) on tendon-bone healing after cruciate ligament reconstruction, and to explore the effect of highly ag-glutinative staphylococcin combined with α-calcium sulfate hemihydrate on anterior cruciate ligament（ACL）reconstruction after ACL reconstruction. Methods: Fifty-four New Zealand white rabbits were randomly divided into three groups: blank control group, α-CSH group, and HAS+α-CSH group. In the blank control group, only autologous ACL reconstruction was performed. HAS binds to a-calcium sulfate hemihydrate complex. At different time after operation (4, 8, 12 weeks after operation), 6 experimental rabbits in each group were randomly selected to be sacrificed and perform gross histological observation, cytological outcome of tendon-bone interface, protein analysis of synovial fluid, and biomechanical test of knee joint. Results: HAS+α-CSH can make the tendon-bone interface appear "tide line"-like structure earlier, reduce the production of TGF-α and IL-1β in synovial fluid, increase the area of new bone formation and the maximum tensile load of the reconstructed tendon , maximum load energy and maximum stress result. Conclusion: The combination of highly ag-glutinative staphylococcin and α-calcium sulfate hemihydrate can promote the tendon-bone healing process after cruciate ligament reconstruction.