AbstractThe direct electron transfer (DET) process, which does not require a mediator, is highly advantageous and holds significant potential for application in biosensors. Enzyme‐based biosensors are simple, cost‐efficient, and easy to use. This review highlights key enzymes with redox‐active sites that are suitable for DET processes using various modified electrodes and their applications in biosensors. The review discusses enzyme classification, distinguishing between single‐cofactor and multi‐cofactor enzymes according to their number of prosthetic groups, with a focus on internal electron transfer processes and the role of redox‐active centers. Methods for enzyme immobilization on electrodes are explored with an emphasis on their impact on electron transfer rates in biosensors. A comparison of sensitivity, electron transfer rates, and the linear detection range of molecules (e.g., glucose and lactose) is provided to illustrate the impact of different immobilization techniques on biosensor performance.