This study aimed to determine the optimal dosage range of NT-3 in the soluble form or loaded with chitosan carriers by using NT-3/chitosan carriers to support the survival and proliferation of neural stem cells (NSCs) and induce them to differentiate into desired phenotypes. NSCs were co-cultured with chitosan carriers loaded with different doses of NT-3. As the control, NSCs were cultured in the defined medium, into which were added different doses of NT-3 in the soluble form every day. The ELISA kit was used to study the NT-3 releasing kinetics, which showed that, in the initial co-culture stage from 1 h to 14 weeks, the chitosan carriers loaded with different doses of NT-3 released NT-3 stably and constantly. The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was conducted to measure the cell viability, and the immunocytochemical methods were adopted to quantitatively analyze the phenotypes differentiating from the NSCs. Compared to the 100 ng NT-3 daily addition group (1400 ng over 14 days), the 25 ng, 50 ng and 200 ng NT-3 daily addition group showed dramatically shorter processes length and much lower differentiation percentage from NSCs into neurons. By contrast, the NT-3 (25 ng)-chitosan carriers group had not only higher cell viability, but also similar processes length and differentiation percentage from NSCs into neurons to the 100 ng NT-3 daily addition group. The method developed in this study significantly reduced the NT-3 amount required to support the survival, proliferation and differentiation of NSCs in vitro. Meanwhile, the chitosan carriers used here provided an ideal 3-dimensional scaffold for the adhesion, migration, proliferation and differentiation of NSC and the differentiated cells. Therefore, this method may open a new field for the large-scaled culture and amplification of NSCs in vitro to replace the lost neural cells, meanwhile lower the consumption of neurotrophic factors in the cell transplantation therapy of brain and spinal injury.