N-Alkylated chitosan (NACS) may improve the blood clotting efficiency of chitosan (CS). To study its blood coagulation capability, a series of NACSs with various carbon chain lengths and degrees of substitution (DS) of alkyl groups were synthesized and characterized by FTIR, NMR, elemental analysis, and X-ray diffraction (XRD). The corresponding NACS nanofiber membranes (NACS-NM) were subsequently fabricated by electronic spinning technique. SEM, XRD, DSC, surface area, porosity, contact angle, blood absorption, and mechanical properties were used to characterize the CS-NM/NACS-NM. Moreover, cytotoxicity, coagulation, activated partial thromboplastin time, plasma prothrombin time, thrombin time, and platelet aggregation tests were performed to evaluate the biocompatibility and blood coagulation properties of NACS-NM. The results showed that NACS-NM was not cytotoxic. NACS-NM with DS of 19.25% for N-hexane CS (CS6b), 17.87% for N-dodecane CS (CS12b), and 8.97% for N-octadecane CS (CS18a) exhibited good blood clotting performance. Moreover, NACS-NMs favored the activation of coagulation factors and platelets. In addition, intracellular Ca2+ was not related to platelet activation. The above results suggested that NACS-NM would be an effective hemostatic agent.