Volumetric muscle loss (VML) caused by injury or trauma affects the quality of life directly, due to its high incidence, lingering healing, and prolonged duration. Tissue engineered scaffolds have been widely used in muscle regeneration for patients with massive muscle injury. Melatonin (MLT) is a bioactive substance secreted by the pineal gland, which can promote muscle recovery by inhibiting oxidative stress and inflammation. Herein, a biocompatible scaffold is developed using thiolated hyaluronic acid (HA-SH), collagen I (COL I), and polycaprolactone/melatonin (PCL/MLT) electrospun membranes. This scaffold mimics extracellular matrix (ECM) and architectural features of native muscle, possesses appropriate mechanical property, stiffness and promotes vascularization. It can provide adhesion sites for C2C12 cells and induce their proliferation and differentiation. Moreover, VML rat model is established to evaluate its effect on muscle regeneration. Results demonstrate that this scaffold possesses a practical application for VML.