Tetanus is a non-communicable disease, preventable with vaccination. Despite the implemented vaccination strategy, a certain number of tetanus cases per year continue to occur. The aim of the study was to evaluate the seroprevalence of anti-tetanus antibodies in the Italian population by age, sex and geographical area. To determine the level of tetanus-specific antibodies, an immunoenzymatic assay was used. A total of 3,821 serum samples were collected in the years 2019–20 from healthy subjects aged 6–90 years residing in 13 Italian regions. Overall, 85 % of the tested subjects resulted positive. The rate of subjects protected against tetanus showed a gradual decrease from the younger age groups to the older ones (6–12 years: 93.6 %, 13–24 years: 91.8 %, 25–39 years: 91.0 %, 40–64 years: 78.2 %, ≥ 65 years: 45.3 %); this is particularly evident in the Southern regions and Islands. Moreover, the prevalence of subjects with low protection (<0.1 IU/ml) was significantly higher in the ≥ 65 age group (10.3 %). Males and females’ prevalence showed a significant difference only in the oldest age group (M: 60.8 %, F: 30.4 %). In general, a higher prevalence was observed for Northern (90.8 %) and Central regions (87.3 %) than Southern regions and Islands (80.0 %). These data, compared with epidemiological ones which showed a high number of cases in the elderly, confirmed that the population with lower protection has a greater risk of contracting the disease, demonstrating the need for adequate immunization through both primary vaccination and boosters for all ages and both sexes, in order to provide lifelong protection.