With the consecutive innovation and application of gene sequencing technology, the third generation sequencing (TGS) based on single molecule real time (SMRT) sequencing technology has demonstrated a potential in a variety of applications in basic and clinical studies, including whole genome assembly, detection of gene structure variation, epigenetic research, etc. This review mainly introduces the principle, characteristics and applications of the TGS technology, especially its applications in virus researches. This review also compares the differences between the next generation sequencing (NGS) and TGS, so as to provide a reference for application based selection for sequencing technology.