Background <p>This study sought to investigate the independent and synergistic impacts of fasting blood glucose (FBG) and serum uric acid (SUA) levels on non-alcoholic fatty liver disease (NAFLD) in participants with and without type 2 diabetes mellitus (T2DM).</p> Method <p>A total of 12,430 participants (mean age: 54.34 ± 15.23, 34.34% female) were enrolled through the Health Screening Center of Tianjin Medical University General Hospital. FBG was classified as &lt; 6 mmol/L, 6–7 mmol/L, and ≥ 7 mmol/L. SUA was classified into two categories: normal SUA and hyperuricemia (SUA level ≥ 420 µmol/L for men, ≥ 360 µmol/L for women). T2DM was ascertained through self-reported data. The diagnosis of NAFLD is established via abdominal ultrasound imaging. Logistic regression models and interaction effect models are used for data analysis.</p> Result <p>Of the 12,430 participants, 4846 (38.99%) were diagnosed with NAFLD. In comparison to individuals with FBG &lt; 6 mmol/L and no self-reported T2DM, those with FBG ≥ 7 mmol/L and no self-reported T2DM exhibited the highest prevalence of NAFLD (odds ratio [OR] 2.91, 95% CI 2.16–3.93) following multi-adjusted analysis. In the joint effect analysis of FBG and SUA, FBG ≥ 7 mmol/L and hyperuricemia were linked to a greater prevalence of NAFLD compared to FBG &lt; 6 mmol/L and normal SUA, both in individuals with self-reported T2DM (OR 2.92, 95% CI 1.68–5.05) and those without self-reported T2DM (OR 7.87, 95% CI 3.57–17.34). An additive interaction existed between FBG and SUA regarding NAFLD in individuals without self-reported T2DM (AP 0.488, 95% CI: 0.068–0.909, <i>P</i> = 0.02).</p> Conclusion <p>Elevated FBG levels are associated with NAFLD irrespective of self-reported T2DM status. The concomitant elevation of FBG and SUA levels exhibits a significant correlation with NAFLD, particularly in individuals lacking self-reported T2DM.</p>