Compared to the commonly used second-generation sequencing (NGS), long-read sequencing (LRS) is capable of continuously reading millions of base pairs. This article reviews the recent progress in the applications of single-molecule real-time sequencing technologies, Oxford nanopore sequencing technologies and novel single-molecule LRS technologies. LRS technologies facilitate the detection of genomic variations, including structural variations, copy number changes, gene fusions, etc. Moreover, LRS technologies provide an effective tool for profiling transcriptome and epigenetic modification. LRS can also solve part of the technical bottleneck of the NGS and help to reveal the complexity of biological macromolecule in health and disease conditions, which may provide a new theoretical basis for better understanding the mechanisms of carcinogenesis and malignant progression, subsequently leading to development of novel therapeutic strategies and drugs. Besides, this review outlines the potential advantages of the application of LRS in clinical practice of oncology.