The authors have prepared a platinum nanoparticle-assembled nanoflake-like SnS2 nanocomposite of the type PtNP@SnS2 as a support for immobilization of glucose oxidase (GOx) to obtain an electrochemical glucose biosensor based on direct electron transfer of GOx. Scanning electron microscopy, transmission electron microscopy, X-ray diffraction, electrochemical impedance spectroscopy, static water contact angle measurements, and cyclic voltammetry were used to characterize the nanocomposite and the glucose biosensor. The nanocomposite has a large surface-to-volume ratio, excellent hydrophiliciy, and high electrochemical activity. This promotes the direct electron transfer between GOx and the surface of a glassy carbon electrode (GCE). The GOx immobilized on the nanocomposite displays good bioactivity, and the surface coverage of the modified electrode is 4.15 × 10−11 mol·cm−2. Under optimum conditions and at a working voltage of −0.4 V vs. SCE, the glucose biosensor exhibits a linear response in the 0.1 to 1.0 mM and 1.0 to 12 mM glucose concentration ranges, with a lower detection limit of 2.5 μM (at an S/N ratio of 3). The biosensor shows excellent selectivity and was successfully applied to the determination of glucose in human serum samples.