Background/Objectives: Antibodies against the hepatitis B surface antigen (anti-HBs) are a marker of immunity against hepatitis B virus (HBV) infections. There is uncertainty about the anti-HBs seroprevalence in the general population of Austria. Methods: We conducted a cross-sectional analysis in blood donors from the Federal State of Tyrol in Austria (August–September 2023) to estimate anti-HBs seroprevalence and median antibody levels. Results: We enrolled 3935 blood donors (median age 47.6 years [25th–75th percentile: 33.3–56.6]; 40.7% female), who were hepatitis B surface antigen negative and had no detectable HBV-DNA. Overall seroprevalence was 51.4% (95% CI: 49.8–52.9%). Anti-HBs seropositivity decreased with higher age (p < 0.001), with 70.3% (66.1–74.3%) being seropositive among participants < 25 years of age and 30.2% (24.2–36.9%) in those aged ≥ 65 years. More females than males were seropositive (54.3% [51.8–56.7%] vs. 49.4% [47.4–51.4%]; p = 0.003). Seroprevalence was significantly higher in urban than in rural areas in participants aged 40 to <55 (p = 0.045) and ≥55 years (p = 0.001). Among 2022 seropositive participants, the overall median anti-HBs antibody level was 539.3 IU/L (25th–75th percentile: 116.3–5417.0). Furthermore, 5% of the participants had an anti-HBs antibody level between 10 and <20 IU/L, 18% between 20 and <100 IU/L, and 77% ≥100 IU/L. Conclusions: Anti-HBs seroprevalence in blood donors from Tyrol, Austria, was 51.4% between August and September 2023 and differed across age, sex, and residence area. Catch-up vaccination programs, especially targeting the elderly living in rural areas, are needed to close HBV immunity gaps.