Cellular heterogeneity of any tissue or organ makes it challenging to identify and study the impact and the treatment of any disease. In this context, analysis of cells at an individual level becomes highly relevant for throwing light on the heterogeneous nature of cells. Single cell analysis can be used to gain insights into an overall view of any disease, thereby holding great applications in health diagnosis, disease identification, drug screening, and targeted delivery. Various conventional methods, such as flow cytometry, are used to isolate and study single cells. Still, these methods are narrower in scope due to certain limitations, including the associated processing/run times, the economy of reagents, and sample preparation. Microfluidics, an emerging technology, overcomes such limitations and is now being widely applied to develop tools for the isolation, analysis, and parallel manipulation of single cells. This review systematically compiles various microfluidic tools and techniques involved in single cell investigation. The review begins by highlighting the applications of microfluidics in single cell sorting and manipulation, followed by emphasizing microfluidic platforms for single cell analysis, with a specific focus on optical sensing-based detection in a high-throughput fashion, and ends with applications in cancer cell studies.