RNA sequencing (RNA-Seq) technology has become a cornerstone in molecular biology and genomics research due to its ability to provide comprehensive insights into gene expression and regulation. This review explores the advancements in RNA-Seq technology, focusing on novel sequencing platforms, increased throughput, and enhanced accuracy. The emergence of singlecell RNA sequencing (scRNA-Seq) and its impact on studying cellular heterogeneity and gene expression at the individual cell level are also discussed. Additionally, the application of longread RNA sequencing in characterizing alternative splicing events and identifying novel transcript isoforms is highlighted. The review also addresses the essential role of integrative bioinformatics tools in effectively analyzing the vast amounts of RNA-Seq data generated. Lastly, the article touches upon the promising and emerging applications of RNA sequencing in biomedical research.