High levels of blood glucose are always associated with numerous complications including cholesterol abnormalities. Therefore, it is important to simultaneously monitor blood glucose and cholesterol levels in patients with diabetes during the management of chronic diseases. In this study, a glucose dehydrogenase from Aspergillus oryzae TI and a cholesterol oxidase from Chromobacterium sp. DS-1 were displayed on the surface of Saccharomyces cerevisiae, respectively, using the yeast surface display system at a high copy number. In addition, two whole-cell biosensors were constructed through the immobilization of the above yeast cells on electrodes, for electrochemical detection of glucose and cholesterol. The assay time was 8.5 s for the glucose biosensors and 30 s for the cholesterol biosensors. Under optimal conditions, the cholesterol biosensor exhibited a linear range from 2 to 6 mmol·L⁻¹. The glucose biosensor responded efficiently to the presence of glucose at a concentration range of 20–600 mg·dL⁻¹ (1.4–33.3 mmol·L⁻¹) and showed excellent anti-xylose interference properties. Both biosensors exhibited good performance at room temperature and remained stable over a three-week storage period.