The transcriptome is the complete set of transcripts for certain type of cells or tissues in a specific developmental stage or physiological condition. Transcriptome analysis can provide a comprehensive understanding of molecular mechanisms involved in specific biological processes and diseases from the information on gene structure and function. Transcriptome has been challenging due to the efficient and fast procedures of RNA-seq. RNA-seq, refers to the use of high-throughput sequencing technologies to sequence cDNA library transcribed from all RNAs in tissues or cells, can be used to quantify, profile, and discover RNA transcripts by sequence reads. Thus, the transcripts can then be mapped on the reference genome to get comprehensive genetic information, such as transcription localization and alternative splicing status. RNA-Seq has been widely used in biological, medical, clinical and pharmaceutical research. The detailed principles, technical characteristics and applications of RNA-seq are reviewed here, and the challenges and application potentials of RNA-seq in the future are also discussed. This will present the useful information for other researchers.