Abstract: In order to improve the extraction rate and utilization rate of collagen in sheepskin, sheepskin collagen was extracted by acid enzyme compounding method, and collagen-based nanofibers were prepared by electrospinning technology. Taking the extraction rate of sheepskin collagen as the evaluation index, the effects of four factors, namely material-liquid ratio, acetic acid concentration, pepsin concentration and enzymatic hydrolysis time, on the extraction effect of sheepskin collagen were investigated, and the optimal level of single factors were determined. On this basis, the process conditions of sheepskin collagen extraction were optimized by orthogonal experimental design. The effects of enzymatic hydrolysis on the structural properties of collagen were explored by biochemical techniques such as ultraviolet spectrum scanning, infrared spectrum scanning, SDS-PAGE mapping and scanning electron microscopy. Collagen and polylactic acid were then compounded and electrospun to obtain collagen-based nanofibers. The results showed that the best process for extracting sheepskin collagen by acid enzyme compounding method was as follows: Material-liquid ratio of 1:25 g/mL, acetic acid concentration of 1.2 mol/L, pepsin dosage of 1.0%, enzymatic hydrolysis time of 72 h. Under these conditions, the extraction rate of sheepskin collagen was 38.42%±0.49%. Ultraviolet spectroscopy showed that sheepskin collagen had a maximum UV absorption peak around 230 nm. Infrared spectrum scanning and SDS-PAGE map analysis showed that sheepskin collagen was mainly composed of α₁, α₂ and β three subunit components, which belonged to type I collagen, and the spatial structure of collagen was intact. Scanning electron microscopy showed that the fiber network structure of sheepskin collagen was relatively intact. The collagen-based nanofibers obtained by electrospinning had a diameter of 418.02±183.77 nm, a tensile strength of 3.616±0.386 MPa, and an elongation at break of 8.69%±1.95%, which had excellent physical properties and the potential to be used as a cell scaffold. This study would provide a theoretical basis for improving the high-value utilization of sheepskin and sheepskin collagen, and also provide theoretical support for the development of collagen-based nanomedical fiber products.