The aim of this study was to determine the prevalence of diphtheria by a cross-sectional in Saudi population. The size of the population consisted of five-hundred healthy subjects from the western regions of Saudi Arabia from six months to 96 years age. ELISA assay kits was used to titers anti-diphtheria IgG. According to the widely used criteria, 31.7% of the total population was susceptible to diphtheria (IgG level <0.15 IU/ml), 15.7% had basic protection (0.15–1.0 IU/ml), and 52.6% had full protection (>1.0 IU/ml). The majority (82%) of the population from 6 months to 96 years had a protective level of IgG against diphtheria. The frequencies of susceptibility were relatively high in middle-aged periods (30.2 – 37.5% of subjects aged 21–50 years). Significantly, more females (20.7%) than males (15%) were unprotected against diphtheria. In conclusion, monitoring immunization status and administering the diphtheria vaccine as required are essential to ensure adequate and long-lasting antibody levels.