Objective To investigate the relationship between serum omega-3 polyunsaturated fatty acid (ω-3PUFA) and insulin resistance(IR) in patients with type 2 diabetes mellitus and non-alcoholic fatty liver disease (NAFLD). Methods This trial involved 51 patients of type 2 diabetes mellitus with NAFLD (G4 group), 50 patients of type 2 diabetes alone(G3 group), 45 patients of NAFLD alone (G2 group) and 42 healthy control subjects (G1 group). Serum ω-3PUFA profile was analyzed with capillary gas chromatography. Insulin resistance was assessed by homeostasis model assessment (HOMA-IR). ALT, AST, γ-glutamyltransferase(GGT) and serum lipids were measured. Results The levels of HOMA-IR were higher in G4 group than those in G3, G2 and G1 group(4. 90 ± 2. 54 vs 2. 38 ± 1.23, 2. 20 ± 1.15, 1.13± 0.42;P<0.05). The level of ALT, AST, GGT, TC, TG, LDL-C were higher in G4 group than those in G3, G2 and G1 group(P < 0. 05). The level of ω-3PUFA was signifieantly lower in G4 group than those in G3, G2 and G1 group(5.68 ±2.02 vs 7. 17 ±2. 38, 6.97±2.32, 10.08±2.76;P <0.05). ω-3PUFA cocentration was negatively correlated with HOMA-IR, TC, TG and LDL-C (r = -0.491, -0. 376, - 0. 462, - 0. 408, P < 0. 05). Conclusions Serum ω-3 PUFA is significantly decreased in patients with type 2 diabetes mellitus and NAFLD. Serum ω-3PUFA is negatively correlated with insulin resistance. ω-3PUEA plays a very important role in the development of diabetes mellitus and NAFLD.