Nucleic acid detection offers critical information in diagnosing pathogen infection and monitoring disease treatment. Point-of-care testing (POCT), with ideal characteristics such as rapidness, user-friendly, sensitivity, specificity, and robustness, has drawn wide attention, especially in resource-poor settings where lack the healthcare infrastructure. Accompanied by the development of microfluidic technologies and new amplification methods, POCT of nucleic acids greatly improves health monitoring and prevents outbreak of infectious diseases. This review summarizes recent advances in diagnostics of nucleic acids in POC formats, and discusses challenges associated with POCT of nucleic acids.