Objective In order to standardize the comet assay, a multilayer electrophoresis tank was invented and validated. Methods Lymphocytes were extracted from ten SD rats. Ten slides were prepared for the lymphocytes of each rat for comet assay. One half slides were treated with 30% H₂0₂, and the other untreated slides were used as negative control. Comet assays on single layer-tank or multi-layer tank were used for comparing the assay results. Results The DNA of lymphocytes was damaged significantly by 30% H₂0₂. There was a significant difference of results between single-layer tank and multilayer tank, but no significant difference of results among different layers of multilayer tank. Conclusion Comparing with the single-layer tank, more samples could be analyzed in multilayer tank, and the comparability of results for comet assay was improved. Multilayer tank would be helpful to standardize comet assays.