<p>As a highly fatal and heterogeneous disease, ovarian cancer (OC) lacks sufficient treatment choices. Over the past decade, RNA sequencing (RNA-seq) has profoundly changed research methods in molecular biology, enabling comprehensive analysis of transcriptomes, somatic mutations, and tumour heterogeneity. In OC research, the application of RNA-seq technologies has significantly enhanced scientists’ understanding of the biological mechanisms underlying the disease, providing deeper insights into the intrinsic mechanisms of key processes such as tumour initiation, metastasis progression, drug resistance formation, and immune evasion. This breakthrough not only expands our understanding of OC but also holds promise for the discovery of novel diagnostic biomarkers, the development of innovative therapies, and the establishment of precise prognostic assessment systems. This review summarizes RNA-seq technologies and their applications in OC research, while also discussing the challenges and opportunities for their broader implementation in cancer studies.</p>