〔Abstract〕 Objective To detect cell DNA damage more effectively by optimizing the influencing factors of Comet assay and establishing a positive control. Methods Based on the traditional alkaline comet assay, the gel preparation method, gel concentration, agarose solvent, lysis time, cell density and electrophoresis voltage were optimized. HL-60 cells were induced by hydrogen peroxide(H2O2) as a DNA damage inducer as a positive control. Results The optimized experimental results showed that the gel preparation method improved from three-step method to two-step method. PBS was used as agarose solvent, 0.8% normal melting point agarose was used to prepare the bottom layer, and 0.7% low melting point agarose was mixed with cell suspension as the second layer. The gel preparation effect was better, the operation was simpler, the time was saved, and the degumming problem was better solved. After lysis for 1 h and electrophoresis at 1 V/cm voltage, representative comet images were obtained. The results were easy to interpret and could effectively avoid false positive results. The positive control of comet assay was successfully established by comparing the DNA damage of HL-60 cells induced by different concentrations of H2O2. Conclusion Compared with the traditional method, the optimized comet assay method is simpler, faster, more accurate, lower cost and has better repeatability, which can quickly detect DNA damage in cells.