Quantum dots (QDs) possess characteristic chemical and optical features. In this light, ZnS QDs capped with glutathione (GSH) were synthesized via an easy aqueous co-precipitation technique. Fabricated QDs were characterized in terms of X-ray diffraction (XRD), high resolution transmission electron microscope (HRTEM), Fourier transform infrared (FTIR) and Zeta potential analyses. Optical properties were examined using photoluminescence (PL) and ultraviolet–visible (UV–visible) spectroscopies. Moreover, GSH-capped ZnS QDs were evaluated as an optical probe for non-enzymatic detection of urea depending on the quenching of PL intensity of ZnS QDs in the presence of urea from concentration range of 0.5–5 mM with a correlation coefficient (R2) of 0.995, sensitivity of 0.0875 mM−1 and LOD of 0.426 mM. Furthermore, GSH-capped ZnS QDs-urease conjugate was utilized as an optical probe for enzymatic detection of urea in the range from 1.0 µM to 5.0 mM. Interestingly, it was observed that urea has a good affinity towards ZnS QDs-urease conjugate with a linear relationship between the change of PL intensity and urea concentration. It was found that R2 is 0.997 with a sensitivity of 0.042 mM−1 for mM concentration (0.5–5 mM) and LOD of 0.401 mM. In case of µM concentration range (1–100 µM), R2 was 0.971 with a sensitivity of 0.0024 µM−1 and LOD of 0.687 µM. These data suggest that enzyme conjugation to capped QDs might improve their sensitivity and applicability. Graphical Abstract