There is an urge in the improvement of mechanical performance of regenerated collagen fibers for their potential biomedical applications such as absorbable surgical sutures. In this work, high-performance collagen fibers and collagen/chitosan composite fibers were prepared by a dry-jet wet spinning method, and the effects of the aspect ratio of the spinneret and air gap length on the structure and mechanical performance of the fibers were investigated. The results showed that when the aspect ratio of the spinneret was 160:1 and the air gap length was 40 mm, the resulting collagen fibers showed excellent mechanical performance with a highly ordered fibrillar microstructure and enhanced structural orientation. Further, the mechanical performance of collagen fibers was improved with the incorporation of chitosan due to their intermolecular hydrogen bond interactions, and the tensile strength of the collagen/chitosan (8:2) composite fibers can reach 178.8 MPa. Moreover, after cross-linking with glutaraldehyde, the tensile strength of the composite fibers was further increased to as high as 198.2 MPa. Benefiting from the natural tissue affinity of collagen and chitosan, the as-prepared bionic collagen/chitosan composite fiber possessed superior wound suture performance and biodegradability in vivo, which provides a new perspective for their potential use in the field of multifunctional biomedical materials in a sustainable way.