Chitosan nanofibers fabricated by electrospinning are contaminated by acidic anions from the acid spinning solution, leading to instability of the nanofibers in aqueous solutions, and the traditional fiber treatment method will also lead to the deterioration of the nanostructure. Here we demonstrate a novel approach to removing the acidic anions with full preservation of the nanofibrous structure. The as-spun nanofibers are first protected (stabilized) by reversible acylation. Second, contaminants are then eliminated by hydrolysis; finally, acylation is reversed. Chemical analysis showed the removal of the acidic anions and the graft and removal of acyl groups. Morphological analysis showed that the reversibly acylated fibers had diameters <150 nm and nanofiber structure was maintained after immersion in aqueous solution. The membranes also were compatible with bone cells in culture. The resultant pure chitosan nanofibers show excellent stability in aqueous solution and exhibit broad potential in biomedical applications.