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Achilles tendon rupture is a severe injury, and its optimal therapy remains controversial. Tissue engineering scaffolds play a significant role in tendon healing and tissue regeneration. In this study, we developed tri-layered doxycycline/collagen/bupivacaine (DCB)-composite nanofibrous scaffolds to repair injured Achilles tendons. Doxycycline, collagen, and bupivacaine were integrated into poly(lactic-co-glycolic acid) (PLGA) nanofibrous membranes, layer by layer, using an electrospinning technique as healing promoters, a 3D scaffold, and painkillers, respectively. After spinning, the properties of the nanofibrous scaffolds were characterized. In vitro drug discharge behavior was also evaluated. Furthermore, the effectiveness of the DCB–PLGA-composite nanofibers in repairing ruptured Achilles tendons was investigated in an animal tendon model with histological analyses. The experimental results show that, compared to the pristine PLGA nanofibers, the biomolecule-loaded nanofibers exhibited smaller fiber size distribution and an enhanced hydrophilicity. The DCB-composite nanofibers provided a sustained release of doxycycline and bupivacaine for over 28 days in vivo. Additionally, Achilles tendons repaired using DCB-composite nanofibers exhibited a significantly higher maximum load-to-failure than normal tendons, suggesting that the biomolecule-incorporated nanofibers are promising scaffolds for repairing Achilles tendons. [ABSTRACT FROM AUTHOR]