Next-Generation Sequencing (NGS) has the range of high-throughput quickly adapted into the countless facet of viral diagnostic research. This method has been extensively applying in full genome sequencing, genetic diversity identification, transcriptomic routine diagnostic work patient care and management, and the new understanding of the interactions between viral and host transcriptome, to promote virus research. An exciting era of viral exploration has begun and will set us new challenges to understand the role of newly discovered viral diversity in both disease and health. In this review, discuss about the preparation of viral nucleic acid templates for sequencing, assay formats underlying next-generation sequencing systems, methods for imaging and base calling, quality control, data processing pipelines and bioinformatics approaches for sequence alignment, variant calling and suggestions for selecting suitable tools. Also discuss of the most important advances that the new sequencing technologies have brought to the fields of clinical virology and challenges behind it.