This paper reports the enhanced performance of cholesterol oxidase (COx) conjugated CdSe/ZnS quantum dots (QDs) by using water-soluble mercaptoacitic acid (MAA) as linker. The functionalized MAA-CdSe/ZnS QDs conjugated in four different dilutions of cholesterol oxidase significantly affected QDs photoluminescence intensities, which affected the process of charge transfer from QDs to MAA. The conjugation of COx to MAA-QDs in increased dilutions resulted in the regain of PL intensities, which were attributed to the passivation of MAA HOMO/LUMO states. The electrochemical impedance spectroscopy and cyclic voltammetry of the conjugated QDs were performed to get study the charge transfer mechanism. The 1:1000 diluted COx conjugated MAA-CdSe/ZnS QDs was found to have the lowest charge transfer resistance of 228 Ω, the highest diffusion (~ 1.39 × 10-13 cm2/s) and charge transfer rates (~ 4.5 × 10-6 s-1) between the electrode and the redox species. The current study demonstrated the sensitivity of electrochemical and optical based detection on the alkaline.