Abstract: The earthworm was used as the research organism. This study used two separate contamination modes (one-time and multiple-time) to simulate the formation of chlorpyrifos accumulating in the soil by different modes of entry into the soil. Starting with the absorption and accumulation of chlorpyrifos by earthworms, this paper studies the genotoxicity of chlorpyrifos to earthworms. The results showed that earthworms were able to absorb and accumulate chlorpyrifos from different contamination modes into the soil, and that chlorpyrifos accumulated in earthworms caused chromosome and DNA damage in the coelom cells of earthworms. Genotoxicity of chlorpyrifos to earthworms aggravates with increasing exposure time and exposure concentration of chlorpyrifos. Earthworms are more prone to uptake and accumulation of chlorpyrifos entering the soil by the multiple-time contamination mode than by the one-time contamination mode, thus showing stronger genotoxicity.