A kind of nanocomposite consisting of molybdenum disulfide (MoS₂) and gold nanoparticles (AuNPs) was prepared by a simple self-reduction method. Transmission electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy were used to characterize the MoS₂@AuNP nanocomposites. The as-prepared MoS₂@AuNP nanocomposites were further used to modify a glassy carbon electrode to construct an electrochemical glucose biosensor. The electrochemical performance of the biosensor was evaluated using cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry. And it was found that the biosensor had prominent electrochemistry activity towards glucose. Under the optimized experimental conditions, a linear relationship in the range of 1–100 μM was detected between the difference of cathodic reduction current and the concentration of glucose, and the detection limit was 0.14 μM. The results showed that the biosensor exhibited good specificity, repeatability and reproducibility. In addition, the biosensor was also successfully applied for the determination of glucose concentrations in milk samples.