Fibroblast growth factor 21 (FGF21) serves as an essential biomarker for early detection and diagnosis of nonalcoholic fatty liver disease (NAFLD). It has received a great deal of attention recently in efforts to develop an accurate and effective method for detecting low levels of FGF21 in complex biological settings. Herein, we demonstrate a label-free, simple and high-sensitive field-effect transistor (FET) biosensor for FGF21 detection in a nonaqueous environment, directly utilizing two-dimensional molybdenum disulfide (MoS) without additional absorption layers. By immobilizing anti-FGF21 on MoS surface, this biosensor can achieve the detection of trace FGF21 at less than 10 fg mL. High consistency and satisfactory reproducibility were demonstrated through multiple sets of parallel experiments for the MoS FETs. Furthermore, the biosensor has great sensitivity to detect the target FGF21 in complex serum samples, which demonstrates its great potential application in disease diagnosis of NAFLD. Overall, this study shows that thin-layered transition-metal dichalcogenides (TMDs) can be used as a potential alternative platform for developing novel electrical biosensors with high sensitivity and selectivity.