The rapid and accurate detection of pathogenic microorganisms is essential for controlling infectious diseases and protecting public health. Traditional methods, including microbial culture, immunoassays, and polymerase chain reaction (PCR), are limited by long turnaround times, cross-reactivity, and reliance on complex instrumentation. In recent decades, DNA frameworks have emerged as promising platforms for constructing high-performance biosensors, owing to their programmable configurations, nanoscale addressability, and dynamic responsiveness. This review summarizes the development of structural DNA nanotechnology and the functionalization strategies of DNA frameworks. We categorize major detection methods empowered by DNA frameworks into electrochemical, fluorescence, and point-of-care testing (POCT) approaches. Their practical applications in clinical diagnosis and environmental monitoring are summarized. Finally, we discuss emerging detection methods and the key challenges that remain to be addressed.