A large proportion of the Chinese population is now at risk of non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the relationship between plasma phospholipids (PL) fatty acids and the risk of NAFLD. One hundred NAFLD patients and 100 healthy subjects were recruited in Hangzhou, China. Plasma PL and selected biochemical and hematological parameters were analyzed by using standard methods. Stepwise logistic regression was used to identify independent risk factors of NAFLD. Plasma PL total saturated fatty acid (SFA), C20:3n-6, serum alanine aminotransferase, high-density lipoprotein cholesterol, and body mass index were independent risk factors of NAFLD. The risk of NAFLD was significantly increased with higher quartiles of plasma PL total SFA (P for trend = 0.028) and C20:3n-6 (P for trend <0.001); plasma PL docosahexaenoic acid (C22:6n-3) was significantly lower in NAFLD patients than in controls (P = 0.032) and the OR of NAFLD in the highest quartile of C22:6n-3 was 0.41 (95 % CI = 0.17-0.97) compared with the lowest quartile. In conclusion, plasma PL total SFA and C20:3n-6 are positively correlated with the risk of NAFLD, while C22:6n-3 is negatively correlated with the risk of NAFLD.