<p>This study developed an enzymatic glucose biosensor based on MoS₂ nanoflower/Ag nanoparticle (MoS₂NF/AgNP) nanocomposites for glucose detection. The nanocomposites were synthesized by a simple chemical method and directly deposited onto a Pt electrode. Glucose oxidase was immobilized on the modified Pt electrode by cross-linking. The structure and morphology of MoS₂NFs and AgNPs were characterized using x-ray diffraction, field-emission scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and ultraviolet visible spectroscopy. Electrochemical properties were investigated by cyclic voltammetry and electrochemical impedance spectroscopy. Results showed that an increased amount of Ag facilitated electron transfer on the sensor’s surface. The biosensor sensitivity levels were 147.46 and 14.36 μA mM⁻¹&#xa0;cm⁻² within 3.5–6.0 and 6.0–11.0&#xa0;mM, respectively. The limits of detection were 66.36 × 10⁻³ and 5.67 × 10⁻³&#xa0;mM within 3.5–6.0 and 6.0–11.0&#xa0;mM, respectively. The effects of experimental parameters including Ag content (wt.%), pH value, enzyme concentration, amount of glutaraldehyde, and scan rate were investigated for optimization. Moreover, the reproducibility, selectivity, and stability were studied for practical application. The developed biosensor was suitable for glucose detection in urine samples, with recovery rates of 93.53–114.92% and relative standard deviation of 1.05–2.55%.</p> Graphical Abstract <p></p>