Nucleic acid testing (NAT) is widely used in disease screening and diagnosis due to its high sensitivity and strong specificity. In addition, whole blood circulation analysis has become a promising non-invasive strategy for cancer diagnosis and surveillance. With the growing testing demand and the advancement of new amplification technologies, nucleic acid detection methods are evolving toward simplicity, speed, and cost-effectiveness. As the gold standard in nucleic acid detection, real-time fluorescence quantitative PCR (qPCR) relies on expensive fluorescence reading equipment and professional operators and is not suitable for the rapid diagnosis of infectious diseases and other diseases. Biosensors have attracted the attention of scientists due to their advantages of rapidity, reliability, and cost-effectiveness. They have been widely applied in medical diagnosis, including point-of-care testing, forensic science, and biomedical research. This paper reviews the recent research progress of NAT methods for pathogenic microorganisms and disease markers and points out future prospects of point-of-care testing, with great significance in improving health care and disease surveillance in resource-constrained areas.