A blood glucose sensor has been developed based on the multilayer films of CdTe semiconductor quantum dots (QDs) and glucose oxidase (GOD) by using the layer-by-layer assembly technique. When the composite films were contacted with glucose solution, the photoluminescence of QDs in the films was quickly quenched because the enzyme-catalyzed reaction product (H2O2) of GOD and glucose gave rise to the formation of surface defects on QDs. The quenching rate was a function of the concentration of glucose. The linear range and sensitivity for glucose determination could be adjusted by controlling the layers of QDs and GOD. The biosensor was used to successfully determine the concentration of blood glucose in real serum samples without sample pretreatment and exhibited satisfactory reproducibility and accuracy.