Aflatoxin B1 (AFB1), a well-established hepatic carcinogen, has limited research on early-stage epigenetic biomarkers for aflatoxin-induced liver damage. In this study, we investigated 168 unpackaged peanut oil (UPP) consumers to evaluate associations among AFB1 exposure, HBV infection, methylation, and liver function. Our findings indicated an average daily AFB1 intake of 3.14 ng/kg·bw/day from UPP oil consumption. The high AFB1 exposure group exhibited significantly elevated gamma-glutamyl transferase (GGT) levels compared with the low AFB1 exposure group ( = 0.030). AFB1 exposure was negatively correlated with methylation status at the 2nd, 8th, and 9th CpG sites of (r = -0.196, -0.192, -0.181, = 0.021, 0.024, 0.036). Furthermore, methylation at the 8th and 9th CpG sites positively correlated with GGT (r = 0.206, 0.203, = 0.019, 0.024). HBV infection significantly influenced methylation, with the HBsAg group exhibiting 16.25% lower methylation ( < 0.05). Stratified analysis by HBV and AFB1 revealed that in the low AFB1 exposure subgroup, methylation in the HBsAg group exhibited a significant 26.38% reduction compared with the HBsAg group. These results indicated that AFB1 and HBV independently and synergistically promote site-specific hypomethylation. Our results implicated methylation as a critical mediator in HBV-AFB1 co-exposure hepatotoxicity, potentially serving as a novel epigenetic biomarker for early liver damage detection.