RNA-seq is a transcriptome analysis method using deep sequencing technology, which uses high-throughput next-generation sequencing technology to investigate, characterize and quantify transcriptome.It can quickly and comprehensively detect almost all transcripts and gene sequences of specific cells in specific tissue of a species, and has been widely used in basic research, clinical diagnosis and drug development and other fields.Its measurement accuracy has been confirmed comparable to microarray and quantitative polymerase chain reaction by several studies.RNA-seq technology can accurately detect mRNA and non-coding RNA which play important roles in the pathogenesis of diabetic retinopathy (DR). Using this technology to investigate the gene regulation and molecular mechanism of DR is helpful to find biomarkers for early diagnosis and prognosis of DR, to comprehensively and systematically study and analyze the molecular mechanism of specific biological process, and to find new therapeutic targets.The application of RNA-seq will be of great significance for the basic research and clinical treatment of DR.In this article, RNA-seq technology was comprehensively and systematically expounded from three aspects, which were the advantages of RNA-seq technology, the selection of sequencing platform as well as library preparation and data analysis.The progress of this technology in DR research was summarized and analyzed.