We report the preparation of poly (propylene imine) dendrimer (PPI) and CdTe/CdSe/ZnSe quantum dots (QDs) as a suitable platform for the development of an enzyme-based electrochemical cholesterol biosensor with enhanced analytical performance. The mercaptopropionic acid (MPA)-capped CdTe/CdSe/ZnSe QDs was synthesized in an aqueous phase and characterized using photoluminescence (PL) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), X-ray power diffraction (XRD), energy dispersive X-ray (EDX) spectroscopy. The absorption and emission maxima of the QDs red shifted as the reaction time and shell growth increased, indicating the formation of CdTe/CdSe/ZnSe QDs. PPI was electrodeposited on a glassy carbon electrode followed by the deposition (by deep coating) attachment of the QDs onto the PPI dendrimer modified electrode using 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) as a coupling agent. The biosensor was prepared by incubating the PPI/QDs modified electrode into a solution of cholesterol oxidase (ChOx) for 6 h. The modified electrodes were characterized by voltammetry and impedance spectroscopy. Since efficient electron transfer process between the enzyme cholesterol oxidase (ChOx) and the PPI/QDs-modified electrode was achieved, the cholesterol biosensor (GCE/PPI/QDs/ChOx) was able to detect cholesterol in the range 0.1–10 mM with a detection limit (LOD) of 0.075 mM and sensitivity of 111.16 μA mM−1 cm−2. The biosensor was stable for over a month and had greater selectivity towards the cholesterol molecule.