Single-walled carbon nanotubes (SWCNTs) feature extraordinary electrical, optical and mechanical properties governed by their unique chiral structures, yet as-synthesized SWCNTs exist as heterogeneous mixtures with severe bundling, which severely restricts their high-performance applications. Over recent decades, tremendous progress has been achieved in the dispersion, chiral sorting and optical sensing of SWCNTs. In this review, we first comprehensively summarize the mainstream dispersion strategies of SWCNTs, including physical dispersion relying on external mechanical forces and non-destructive chemical dispersion such as polymer wrapping, DNA wrapping, surfactant adsorption and aromatic small molecule π–π stacking. Subsequently, we systematically introduce several representative chiral sorting techniques, namely aqueous two-phase extraction (ATPE), density gradient ultracentrifugation (DGU), gel column chromatography (GCC), and polymer-based wrapping approach. Furthermore, we highlight the research advances of SWCNT-based near-infrared (NIR) fluorescent sensors for the detection of metal ions, neurotransmitters, nucleic acids, proteins and viruses, as well as their applications in in vivo biological detection of plants and animals. Finally, we discuss the existing challenges in the scalable preparation of chirality-pure SWCNTs, sensor stability and in vivo applications, and prospect the future development directions toward practical translation. This review aims to provide a systematic reference for promoting the development and application of SWCNTs in advanced optical biosensing.