Acetone is a dangerous material that poses a major risk to human health. To protect against its harmful impacts, a fluorescent biosensor 3-aminopropyl triethoxysilane capped ZnO quantum dots (APTES/ZnO QDs) was investigated to detect low concentrations of acetone. Numerous techniques, including Fourier transform infrared (FTIR), energy dispersive X-ray (EDX), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), zeta potential, UV-vis absorption, and photoluminescence (PL), are used to thoroughly verify the successful synthesis of pristine ZnO QDs and APTES/ZnO QDs. The HRTEM micrograph showed that the average size distributions of ZnO QDs and APTES/ZnO QDs were spherical forms of 2.6 and 1.2 nm, respectively. This fluorescent probe dramatically increased its sensitivity toward acetone with a wide linear response range of 0.1–18 mM and a correlation coefficient (R2) of 0.9987. The detection limit of this sensing system for acetone is as low as 42 μM. The superior selectivity of acetone across numerous interfering bioanalytics is confirmed. Reproducibility and repeatability experiments presented relative standard deviations (RSD) of 2.2% and 2.4%, respectively. Finally, this developed sensor was applied successfully for detecting acetone in a diabetic patient's urine samples with a recovery percentage ranging from 97 to 102.7%.