With the development of DNA sequencing techniques, the next-generation sequencing (NGS) techniques with the characteristics of high-throughput and low cost have become the first choice for more and more researchers to carry out the biological researches. Among the next-generation sequencing techniques, the 454 sequencing platform is the first commercially available and relatively mature one and widely used in various fields of biological research. Taking 454 sequencing platform as an example, we illustrate the advantages and disadvantages of NGS technical principles, review their applications in plant transcriptome, and outlook their future development and applications in plant research field.