The cumulative effects of hepatic injury due to hepatitis B virus (HBV) infections and aflatoxin-B1 (AFB1) exposure are the major risk factors of HCC. Understanding early metabolic changes involving these risk factors in an animal model closely resembling human hepatocellular carcinoma (HCC) is critical for biomarker discovery and disease therapeutics. We have used the hepatitis B surface antigen (HBsAg) transgenic mouse model that mimics HBV carriers with and without AFB1 treatment. We investigated early metabolic changes from preneoplastic state to HCC by non-invasive longitudinal imaging in three HCC groups of mice: HBsAg + AFB1(Gp-I), AFB1 alone (Gp-II), HBsAg alone (Gp-III) and a control group (wild-type untreated; Gp-IV). For the first time, we have identified acylcarnitine signals in vivo in the liver prior to the histological manifestation of the tumors in all three groups. Acylcarnitine concentration increased with increase in tumor growth in all HCC mouse models, indicating elevated metabolic activity and increased cell turnover. This was confirmed in a pilot study using human serum from HCC patients, which revealed a higher concentration of acylcarnitine compared with normal subjects. Translational clinical studies can be designed to detect acylcarnitine in patients with high risk factors for HCC.