Azamethiphos (AZA), an organophosphate pesticide, is well-known for its cholinesterase inhibition and associated toxic risks to non-target organisms. Its high-water solubility facilitates environmental contamination and persistence, increasing the risk of human exposure through bioaccumulation in agricultural products. This study investigates AZA's DNA-binding potential and underlying interaction mechanisms. Using in silico techniques, we analyzed AZA's interactions with DNA, revealing that hydrogen bonding plays a crucial role in stabilizing the AZA-DNA complex. The study found that AZA preferentially binds to AT-rich regions of Ct-DNA, suggesting it acts as a groove binder by fitting into the grooves of the DNA double helix Additionally, fluorescence spectroscopy studies of AZA with DNA were conducted at three temperatures (288 K, 298 K, and 308 K). These experiments demonstrated that AZA binds to Ct-DNA with a moderate binding affinity (3.868, 2.238 and 0.0061 x 10