(1) Background: Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease caused by the destruction of pancreatic insulin-producing beta cells. T1D is one of the most common endocrine and metabolic disorders occurring in children. Autoantibodies against pancreatic insulin-producing beta cells are important immunological and serological markers of T1D. Zinc transporter 8 autoantibody (ZnT8) is a recently identified autoantibody in T1D; however, no data on ZnT8 autoantibody in the Saudi Arabian population have been reported. Thus, we aimed to investigate the prevalence of islet autoantibodies (IA-2 and ZnT8) in adolescents and adults with T1D according to age and disease duration. (2) Methods: In total, 270 patients were enrolled in this cross-sectional study. After meeting the study’s inclusion and exclusion criteria, 108 patients with T1D (50 men and 58 women) were assessed for T1D autoantibody levels. Serum ZnT8 and IA-2 autoantibodies were measured using commercial enzyme-linked immunosorbent assay kits. (3) Results: IA-2 and ZnT8 autoantibodies were present in 67.6% and 54.6% of patients with T1D, respectively. Autoantibody positivity was found in 79.6% of the patients with T1D. Both the IA-2 and ZnT8 autoantibodies were frequently observed in adolescents. The prevalence of IA-2 and ZnT8 autoantibodies in patients with a disease duration < 1 year was 100% and 62.5%, respectively, which declined with an increase in disease duration (p< 0.020). Logistic regression analysis revealed a significant relationship between age and autoantibodies (p< 0.004). (4) Conclusions: The prevalence of IA-2 and ZnT8 autoantibodies in the Saudi Arabian T1D population appears to be higher in adolescents. The current study also showed that the prevalence of autoantibodies decreased with disease duration and age. IA-2 and ZnT8 autoantibodies are important immunological and serological markers for T1D diagnosis in the Saudi Arabian population.