Recent developments in electrochemical biosensors have resulted in new applications in a variety of disciplines, such as clinical diagnosis, food processing quality control, and environmental monitoring. The electrochemical biosensor is a widely used sensing device that transmits an electrical signal from biological activities. Electrochemical biosensors have led to significant advancements in detecting various biomolecules such as uric acid, glucose, cholesterol, lactate, and DNA, including cancer biomarkers, viruses, and antibodies. An electrode is a vital part of this kind of sensor since it serves as a strong support for immobilizing biomolecules (enzyme, antibody, and nucleic acid) and allowing electron flow. Redox enzymes are the foundation of enzymatic electrochemical biosensors, which use oxidation or reduction reactions to detect a substrate, which is then converted into an electrical signal. Immobilizing enzymes onto solid supports is a common method to enhance the performance of an enzymatic electrochemical biosensor. However, there are some challenges, such as potential loss of enzyme activity, difficulties in maintaining enzyme stability, and issues related to the reproducibility and consistency of enzyme immobilization. This review covers all challenges in enzymatic electrochemical biosensors. Numerous new biosensor platforms are announced each year as researchers look for various electrode materials. Additionally, the researchers should familiarize themselves with the practical methods used in electrode production in order to build a successful biosensor. This review highlights current developments in modifying enzyme-immobilized biosensors for detection of different biomolecules. The article further describes methods of enzyme immobilization and various enzymes with different nanomaterials for the development of novel enzymatic electrochemical biosensors.