Abstract: Transcriptome is an RNA set transcribed by a specific cell or tissue in a specific time or state,which allows to study gene function and structure from the overall level and can well display the number and activity of expressed genes.RNA-Seq,as a new high-throughput sequencing technology for transcriptome analysis,has been widely used in biomedical research,because it can provide organism transcription information more quickly and accurately.As the incidence of gastrointestinal cancer increases worldwide,the RNA-Seq technology has achieved some new progress in whole transcriptome resequencing of gastrointestinal tumors.In this article,we will discuss the principle and advantages of RNA-Seq technology as well as its application in the research of gastrointestinal tumors.