<p>Single-cell RNA sequencing (scRNA-seq) and its integration with multi-omics technologies such as epigenomics and spatial transcriptomics are revolutionizing our traditional understanding of cellular heterogeneity and the microenvironment in the nervous system. While technical reviews abound, translating multi-omics data into biological and clinical insights remains a challenge. This review comprehensively outlines the latest advancements in scRNA-seq technology and its integration with multi-omics approaches and artificial intelligence. We systematically summarize its applications across neuroscience, from unraveling neurodevelopment and evolution to deciphering the mechanisms of neurological diseases such as Alzheimer’s disease, Parkinson’s disease, and gliomas. By deeply resolving cell-specific expression differences in neurological disorders, scRNA-seq has enabled the discovery of novel cell subtypes and revealed intercellular regulatory mechanisms, thereby facilitating the deconstruction of disease pathogenesis and the identification of new potential therapeutic targets. Furthermore, this technology has demonstrated significant value in drug screening, efficacy evaluation, and the development of novel treatment strategies. However, scRNA-seq still faces multiple technical limitations. Future efforts should focus on reducing its application costs, addressing clinical ethical concerns, and progressively advancing the clinical translation of scRNA-seq technology.</p>