The persistence of extracellular DNA (eDNA) is crucial for ensuring species diversity and ecological function in aquatic systems. However, scarce information exists about the impact of pesticides on eDNA, although they often co-exist in the aquatic environment. Using a variety of spectroscopic analyses, eDNA degradation and the associated alterations in DNA secondary structure was investigated by exposing DNase I to tested DNA in the presence of chlorpyrifos, a commonly used organophosphate pesticide. Molecular dynamics simulation was used to explore the weak interactions between the tested DNA and chlorpyrifos. The results indicated that chlorpyrifos significantly enhanced DNA degradation without affecting the enzyme activity of DNase I in an aqueous system. Spectroscopic experiments confirmed that chlorpyrifos and the analog chlorpyrifos-methyl could bind with DNA to cause the bases noncovalent stacking interaction. Molecular simulations further demonstrated that pesticide binding with DNA molecules caused widening of the DNA grooves and destruction of the hydrated layer, which enhanced DNA degradation. The findings presented herein provide novel insight into the genotoxicity and ecotoxicity of chlorpyrifos and chlorpyrifos-methyl, as well as their impacts on DNA persistence in aquatic environments.