Endosulfan, as one of the most widely used organochlorine pesticides in the world, has increased the public concern about genotoxicity in soil ecosystems. The comet assay has been widely used in the fields of genetic toxicology and environmental biomonitoring. In the present study we conducted comet assay of endosulfan in earthworm (Eisenia foetida) and white clover (Trifolium repens L.), which are sensitive organisms suitable for acting as a bioindicator for agricultural ecosystems. Earthworms were exposed to endosulfan concentrations of 0.1, 1.0, and 10.0 mg/kg in the soil. White clover roots were immersed in hydroponic pots containing nutrient solutions of different endosulfan concentrations: 0.1, 1.0, and 10.0 mg/L. Tissues from each treatment were collected on the 7th, 14th, 21st, and 28th days of treatment process. Significant effects (p < 0.01) of both concentrations and times of exposure were observed. And endosulfan induced DNA damage in earthworm and white clover nuclei. The comet assay can be used as a reliable tool for early detection of endosulfan.