Coastal marine areas are frequently contaminated by pollutants from fishing vessels, oil spills, and other sources. Litopenaeus vannamei prawns were collected from estuaries and coastal marine areas near mangrove ecosystems that act as pollutant conduits. To limit pollution, animals use defensive systems with heightened enzyme levels. Enzymes like hepatic cytochrome P450-associated enzymes are used as biomarkers. A study assessed the capacity and function of certain enzymes in the hepatopancreas microsomal fractions of Litopenaeus vannamei. The study collected samples from three locations: Kasimedu fishing harbor, Rayapuram, Chennai, a moderately polluted offshore site near mangrove areas, and the least oil-polluted site, Velar estuary, Parangipettai mangrove. The majority of the pollutants is polycyclic aromatic hydrocarbons (PAHs) due to the halting of fishing vessels and other man-made activities. Cytochrome P450 isoforms (CYP450) play the important role of defense mechanism against the PAHs. In this study, we measured EROD (CYP1A1), MROD (CYP1A2), and BROD (CYP2B6) levels from hepatopancreas microsomal fractions of Litopenaeus vannamei. The mixed-function oxygenases (MFOs), also known as monooxygenases or hydroxylases, showed a hierarchical pattern of activities in response to oil contamination, with greater activity in highly oil-contaminated sites (Kasimedu Station-1 and Station-2) compared to the low oil-contaminated reference area (Vellar estuary). The hepatic tissues showed enhanced MROD activity, which strongly correlates with hydrocarbon contamination levels, making it a reliable biomarker for detecting petroleum hydrocarbons in Litopenaeus vannamei. Antioxidant enzymes like superoxide dismutase, catalase, and glutathione reductase increased in activity, indicating their potential as antioxidants, which neutralize reactive oxygen species (ROS). Among the enzymes analyzed, the MROD activity was best correlated with the level of hydrocarbon contamination. Therefore, MROD can be considered a robust biomarker for petroleum hydrocarbons in Litopenaeus vannamei