Tendon injury is common in sports and other rigorous activities, which may result in dysfunction and disability. Recently, scaffolds with a knitted structure have been widely applied for tendon tissue engineering. The purpose of this study was to fabricate a novel knitted tendon scaffold made of microfiber/nanofiber core-sheath yarns and evaluate the biocompatibility and the effect of tenogenic differentiation and tendon tissue regeneration in vitro and in vivo. Poly(ε-caprolactone) (PCL) microfibers, PCL microfibers-PCL nanofibers (PCL-PCL) and PCL microfiber-silk fibroin/poly(l-lactic acid-co-ε-caprolactone) nanofiber (SF/PLCL) core-sheath yarns were fabricated and then knitted with an automatic knitting machine to produce PCL, PCL-PCL and PCL-SF/PLCL fabric scaffolds. The characterization of the scaffolds was performed by using scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy and an universal mechanical instrument. The in vitro experiment showed that rabbit bone marrow stem cells seeded on the scaffolds exhibited an elongated morphology and proliferated better in the PCL-SF/PLCL group, as compared to the PCL and PCL-PCL groups. Moreover, the PCL-SF/PLCL scaffold promoted the tenogenic differentiation of the cells for the highest expression levels of the tendon-related genes through down-regulating p-ERK1/2 expression among the three groups. Furthermore, the in vivo study in a rabbit patellar defect model demonstrated that the PCL-SF/PLCL scaffold could enhance the tissue regeneration and remodeling process as indicated by the better structural and biomechanical properties according to the results of histology, immunohistochemistry, transmission electron microscope examination and biomechanical tests. Therefore, the PCL-SF/PLCL scaffold is proved to be a promising biomaterial for tendon tissue engineering and a potential candidate for clinical treatment of tendon injury in the future.