Neural stem cells (NSCs) in the adult central nervous system play essential roles in both normal homeostasis and repair of damaged tissue after injury. The study of adult NSCs is hampered by the heterogeneous NSC population. In this review, we describe recent progresses in using single-cell RNA-sequencing (scRNA-seq) technique for the investigation of NSCs. The first part of this review focuses on the scRNA-seq techniques and bioinformatic analysis. The second part emphasizes the applications of scRNA-seq analysis in NSC research. Finally, we discuss the challenges and future directions of scRNA-seq technique for both basic research and regenerative medicine.