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Aim: The association between sugar‐sweetened beverages and metabolic disorders has been well studied. However, it has not been determined whether fasting serum fructose is associated with metabolic dysfunction‐associated fatty liver disease (MAFLD). Methods: Participants were enrolled from 2011 to 2012 in Shanghai. Fasting serum fructose concentration was measured with a validated liquid chromatography–tandem mass spectrometry method. Results: A total of 954 participants without diabetes were included. They were followed for an average of 3.5 years. A total of 320 (33.5%) participants had MAFLD at baseline. With the increase in fasting serum fructose level by quartile, the MAFLD prevalence was increased by 27.0%, 25.0%, 37.4%, and 44.5%, respectively (p < 0.001). Each SD increase in fasting serum fructose level was associated with a 60% increased risk of MAFLD (odds ratio 1.60; 95% confidence interval [CI], 1.36–1.88; p < 0.001). Fasting serum fructose levels were more closely associated with four components of MAFLD (hepatic steatosis, prediabetes, insulin resistance, and low high‐density lipoprotein). We built a diagnostic model named the fructose fat index (FFI). The area under the receiver operating characteristic curve of the FFI was 0.879 (95% CI, 0.850–0.908) in the derivation cohort and 0.827 (95% CI, 0.776–0.878) in the validation cohort. Subsequent prospective studies found that the incidence risk of MAFLD was 2.26 times higher in the high‐fructose group than in the low‐fructose group among female participants (95% CI, 1.46–3.49; p < 0.001). Conclusion: Fasting serum fructose concentration, which mostly reflects endogenous fructose, was associated with a higher risk of MAFLD. The FFI derived from fasting serum fructose could be used to predict MAFLD. [ABSTRACT FROM AUTHOR]