There are many mysteries of the nervous system and neurodegenerative diseases. Evidences showed that neural stem cells (NSCs) play a crucial role in recovering nervous system, although this was not sufficient due to lack of cell number. This research was carried out to isolate and culture NSCs from murine foetal brain and our NSCs were cultured as floating and multicellular neurospheres. The neurosphere has been consider as an excellent tool to investigate the differentiation and proliferation of NSCs. Propagation of NSCs in vitro was essential for understanding neural cell’s fate and supplying a promising source for cell therapy for neurodegenerative disease. Serum-free medium with mitogens, N2 and B27 supplement was appropriate for NSC culture. To characterize candidate cells, we assessed stemness and stem cell markers by sphere formation assay, differentiation assay, and immunocytochemistry. The results showed that NSCs could develop as floating spheres; they expressed stem cell-like characteristics and popular marker of neural stem cells-nestin, and differentiate into nerve cells. This population of cells was a promising source to studying neural stem cells and the treatment of neurodegenerative disease.