The organochlorine pesticide dicofol (DCF), a persistent organic pollutant, is used as acaricide worldwide. Considering its large consumption in the agriculture sector and potential toxic effects such as endocrine disruption, carcinogenicity, and environmental persistence are detrimental to human health. To take an extensive evaluation of its potential toxicity, the current study was aimed to explore the binding mechanism and adverse effect of DCF on human serum albumin (HSA) by using an array of biophysical techniques (UV-visible, fluorescence, 3D fluorescence, and circular dichroism spectroscopy), isothermal titration calorimetric (ITC), computational methods and biochemical approaches. Fluorescence quenching and UV-Visible spectra of the HSA-DCF system confirmed static quenching mechanism and complex formation between HSA and DCF. The thermodynamics results from ITC revealed DCF-HSA interaction was exothermic and spontaneous and involved hydrophobic interactions and hydrogen bonding. The esterase activity of HSA displayed constant V and elevated K values confirming DCF-HSA competitive interaction. Circular dichroism spectra results revealed structural changes in HSA protein on interaction with DCF. Furthermore, molecular-specific site marker and molecular modelling results affirmed that the binding Site of DCF is Site I of HSA. A significant carbonyl content level in DCF-HSA system suggested protein structure damage. This work is likely to add a better understanding of DCF toxicity in human health and helpful in fortifying the check on food safety.