Nucleic acid detection (specifically, DNA and RNA analysis) is of great importance to clinical diagnosis, forensic analysis, biological and biomedical fields, and food safety. Because of the increasing number of targets to be detected, traditional single detection techniques cannot fulfill the necessary requirements. Hence, multiple and high-throughput detection methods have been developed. These technologies can save time and reduce costs; moreover, the validation of their results is simpler and more intuitive. This review mainly describes the basic principle as well as the advantages and applications of each commonly used high-throughput detection method.