Electrospun collagen nanofibers are effective for wound healing; however, many problems, such as the tedious preparation process, weak strength and poor structure integration, limit further applications. In this study, recombinant human collagen (RHC) peptides and a simple one-step crosslinking strategy were used to prepare RHC/chitosan nanofibers. With the nonpathogenic, water-soluble RHC and a mild electrospinning solvent, in situ crosslinked nanofibers (S-CN) not only simplified the preparation procedure but also maintained a more integrated morphology. Compared with the immersed crosslinked nanofibers (I-CN), S-CN showed better performance in moisture retention, degradation and mechanical strength tests. In vitro cell proliferation, morphology and RT-PCR studies confirmed that fibroblasts presented better activities on nanofibers crosslinked in situ. Importantly, after treating with the nanofibers, rapid epidermidalization and angiogenesis were observed in an SD rat scalding model. All these data suggest that electrospun RHC/chitosan nanofibers crosslinked in situ are an ideal candidate that can be used for wound healing applications.