Abstract A reusable quartz crystal microbalance (QCM) biosensor based on the toehold-mediated strand displacement reaction (SDR) is first developed for highly specific detection of single-base DNA mutation. N5, N10-methylenetetrahydrofolate reductase (MTHFR) gene C677T mutation is chosen as a model to investigate the performance of the constructed biosensor. The capture DNA immobilized on the gold electrode surface of QCM hybridizes with the conjugate of biotinylated reporter DNA and streptavidin (abbreviated to reporter probe) to form the recognition layer. A toehold domain in the reporter DNA is specifically designed to bind the complementary mutant DNA and trigger the SDR, eventually resulting in the release of the complex of reporter probe and mutant DNA from the gold surface, and then the recognition layer is mildly regenerated by subsequently injecting the reporter probe again. The fabricated biosensor shows good repeatability for the mutant DNA, as more than 87% signal intensity of the sensor remained after six repetitive measurements. A linear range of 1–75nM is achieved with a detection limit of 0.8nM at room temperature. This biosensor also affords remarkable specificity to the mutant DNA against its wild-type DNA with a discrimination factor of 33. The potential of applying this biosensor in screening real samples is confirmed by spiked HeLa cells lysate with a recovery of 108%. The proposed sensing strategy provides a simple and universal solution for repetitive and highly specific detection of single-base DNA mutation.