Assessing bacterial viability is crucial in public health, food safety, environmental microbiology, and other relevant fields. The classical agar plate counting method and the popular dye-based assays have shown their strengths, but they also have limitations including high time consumption, relatively complex sample preparations, and cytotoxicity. In this work, we present a new bacterial viability assay based on optical tweezers utilizing a power sweeping strategy. By monitoring and analyzing bacterial nanomotion in optical traps under different trapping laser powers, the slope of the proportionality between the quantified extent of motion and the trapping laser power was defined as the mobility restriction coefficient (MRC) to quantify bacterial viability. We first established a firm correlation between the viability and MRC by measuring alive and dead Escherichia coli and Photobacterium phosphoreum. Then the capability of real-time long-term characterization of the assay was validated by measuring the viability of individual P. phosphoreum while regulating the viability with an inactivation light. Notably, a 'spinning-induced stabilization' mechanism was proposed to explain the surprising increase of apparent bacterial mobility after inactivation. Overall, the assay was proved to be a reliable label-free bacterial viability assay at a single-cell level, which holds potential in antibiotic susceptibility testing, drug screening, and rapid diagnostics.