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Objective: The purpose of this study was to explore the effect of fibroblast growth factor 2 (FGF-2) on collagenous fibre formation and the osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs) in vitro, as well as the effect of FGF-2–induced hAMSCs combined with autologous platelet-rich plasma (PRP) on tendon-to-bone healing in vivo. Methods: In vitro, hAMSCs were induced by various concentrations of FGF-2 (0, 10, 20, and 40 ​ng/ml) for 14 days, and the outcomes of ligamentous differentiation and osteogenic differentiation were detected by quantitative real-time reverse transcription PCR, Western blot, immunofluorescence, and picrosirius red staining. In addition, a lentivirus carrying the FGF-2 gene was used to transfect hAMSCs, and transfection efficiency was detected by quantitative real time reverse transcription PCR (qRT-PCR) and Western blot. In vivo, the effect of hAMSCs transfected with the FGF-2 gene combined with autologous PRP on tendon-to-bone healing was detected via histological examination, as well as biomechanical analysis and radiographic analysis. Results: In vitro, different concentrations of FGF-2 (10, 20, and 40 ​ng/ml) all promoted the ligamentous differentiation and osteogenic differentiation of hAMSCs, and the low concentration of FGF-2 (10 ​ng/ml) had a good effect on differentiation. In addition, the lentivirus carrying the FGF-2 gene was successfully transfected into hAMSCs with an optimal multiplicity of infection (MOI) (50), and autologous PRP was prepared successfully. In vivo, the hAMSCs transfected with the FGF-2 gene combined with autologous PRP had a better effect on tendon-to-bone healing than the other groups (p ​< ​0.05), as evidenced by histological examination, biomechanical analysis, and radiographic analysis. Conclusion: hAMSCs transfected with the FGF-2 gene combined with autologous PRP could augment tendon-to-bone healing in a rabbit extra-articular model. The translational potential of this article: hAMSCs transfected with the FGF-2 gene combined with autologous PRP may be a good clinical treatment for tendon-to-bone healing, especially for acute sports-related tendon–ligament injuries.