This retrospective study aimed to assess the differences in serum biomarker levels, specifically thyroid hormone (free T4 and Thyroid-Stimulating Hormone [TSH]) levels, according to the presence or absence of Hepatitis B Virus (HBV) infection. Using a quantitative immunofluorescence assay, we analyzed serum samples from patients with confirmed HBV infection at Dankook University Hospital in Cheonan Province, Republic of Korea, between August 2022 and September 2022. The study population was divided into two groups: Young individuals (<60 years) and older adults (≥60 years). Our findings indicated that differences in thyroid hormone levels between HBV-positive and negative participants were not significant. However, when considering age groups, TSH and free T4 levels in young individuals with HBV-positive serum were 1.78±0.09 µIU/mL (normal range: 0.4-5.0 µIU/mL) and 1.24±0.02 ng/mL (normal range: 0.8-1.9 ng/mL), respectively. In older adults with HBV-positive serum, the corresponding values were 2.22±0.17 µIU/mL and 1.24±0.07 ng/mL, respectively. Our results suggest that the influence of HBV on anti-pituitary hormone levels is age-dependent rather than virus related. Furthermore, the quantitative immunofluorescence method used in this study provides a simpler alternative to enzyme-linked immunosorbent assay or electrochemiluminescence immunoassay for assessing serological thyroid hormone levels in patients with HBV. These findings can aid in the interpretation of diagnostic test results and the timely detection of metabolic changes associated with HBV infection.