Background: Leukocyte telomere has been shown to be related to insulin resistance-related diseases, such as type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). This cross-sectional study investigated the association of leukocyte telomere length (LTL) with NAFLD in T2DM patients. Methods: Clinical features were collected and LTL was measured by Southern blot-based terminal restriction fragment length analysis in 120 T2DM patients without NAFLD and 120 age-matched T2DM patients with NAFLD. NAFLD was clinically defined by manifestations of ultrasonography. The correlation between LTL and clinical and biochemical parameters were analyzed by Pearson correlation or Spearman correlation analysis. Factors for NAFLD in T2DM patients were identified using multiple logistic regressions. Results: LTL in T2DM patients with NAFLD were significantly longer than those without NAFLD (6400.2 +/- 71.8 base pairs [bp] vs. 6023.7 +/- 49.5 bp, P < 0.001), especially when diabetes duration was less than 2 years. Meanwhile, the trend of shorter LTL was associated with the increased diabetes duration in T2DM patient with NAFLD, but not in T2DM patients without NAFLD. Finally, LTL (odds ratio [OR]: 1.001, 95% confidence interval [CI]: 1.000-1.002, P = 0.001), as well as body mass index (OR: 1.314, 95% CI: 1.169-1.477, P < 0.001) and triglycerides (OR: 1.984, 95% CI: 1.432-2.747, P < 0.001), had a significant association with NAFLD status in T2DM patients. Conclusions: T2DM patients with NAFLD had a significantly longer LTL than those without NAFLD. The longer LTL was especially evident in the early stage of T2DM, indicating that longer LTL may be used as a biomarker for NAFLD in T2DM patients.