[Objective] To optimize the experiment protocol of ultraviolet (UV) radiation of Bacillus subtilis so that students can get better results in the experiment lesson. [Methods] We optimized the protocol by determining the best bacterial cultivation, estimation of viable cells, and UV-radiation parameters. [Results] Liquid medium was better than agar slant to obtain a homogeneous suspension of cells. The cell amount of the initial bacterial suspension was estimated efficiently by turbidimetric method, which was convenient to dilute the initial suspension to a concentration of 102?103 CFU/mL. Dry surface covered with dry dish was necessary for the formation of singlet colonies on a bacteria-inoculated plate. During UV exposure, the lethal effect was affected by the adding volume of suspension and mixing speed. [Conclusion] The optimized protocol produces repeatable results easily and can be used for teaching or research.