Novel amphipathic derivative of chitosan (carboxymethyl-hexanoyl chitosan, CHC) was made into mats of nanofibers (approximately 100 nm) via electrospinning. The resulting mats were further cross-linked with genipin. The morphology of CHC nanofibers was examined using a field emission scanning electron microscope (FESEM). The optimum parameters of CHC nanofiber was achieved when the CHC concentration was 4 wt% and electrospinning was conducted with a voltage of 20 kV over a distance of 10 cm. The characterizations of biocompatibility, hemocompatibility, and anti-bacterial activity of the nanofibers were also investigated. The results show that CHC nanofibers still preserved antibacterial activity and thrombogeneicity owing to those residual amino groups of chitosan and exhibit high biocompatibility for L929 fibroblast test. Thus CHC exhibited the potential to serve as a novel wound dressing and surgical implants application by these advanced features.