Organic pollutants have become a serious environmental concern due to improper use of agricultural chemicals, mining activities, and large volumes of urban waste discharge. Organic pollutants have emerged as a significant ecological issue because of the misuse of agricultural chemicals, mining operations, and the substantial discharge of urban waste. Phenanthrene (PHE), a polycyclic aromatic hydrocarbon (PAH), has various industrial applications such as dyes, plastics, pesticides, explosives, and drugs. In this study, to estimate the Human serum albumin (HAS) binding and liver cytotoxicity of PHE, the in vivo, in vitro, and in silico investigation was applied. The results show that the interaction between PHE and HSA can be done, spontaneously. The in vivo study demonstrated that both low- and high doses of PAH led to a decrease in hepatocytes and sinusoid volumes and an upward trend in blood vessels compared to the control group. The total measured biochemical factors except for HDL showed an increase in both doses of PAH compared to controls. The serum level of HDL in Phe-treated rats was markedly decreased compared to control rats.