Controlled release of neurotrophic factors to target tissue via microsphere-based delivery systems is critical for the treatment strategies of diverse neurodegenerative disorders. The present study aims to investigate the feasibility of the controlled release of bioactive nerve growth factor (NGF) with ionically cross-linked chitosan microspheres (NGF–CMSs). The microspheres were prepared by the emulsion-ionic cross-linking method with sodium tripolyphosphate (STPP) as an ionic cross-linking agent. The size and distribution of the microspheres, SEM images, Fourier transform infra red spectroscopy (FT-IR), encapsulation efficiency, in vitro release tests and bioactivity assay were subsequently evaluated. We found that the microspheres had relatively rough surfaces with mean sizes between 20 and 31 μm. FT-IR results provided evidence of ionic interaction between amino groups and phosphoric groups of chitosan and STPP. The NGF encapsulation efficiency ranged from 63% to 88% depending on the concentration of STPP. The in vitro release profiles of NGF from NGF–CMSs were influenced by the concentration of STPP. NGF–CMSs which were cross-linked with higher concentration of STPP showed slower but sustained release of NGF. In addition, the released NGF from NGF–CMSs was capable of maintaining the viability of PC12 cells, as well as promoting their differentiation. Taken together, our findings suggest that NGF–CMSs are capable of releasing bioactive NGF over 7 days, thus having potential application in nerve injury repair.