Biosensors have emerged as a potential tool for selective and sensitive detection. Biomolecules like enzymes, deoxyribonucleic acid (DNA), and antibodies are used as recognition sites in biosensors due to their high selectivity, sensitivity, and signal-to-noise ratio. Nowadays, biosensors are used to detect many vital biomolecules such as glucose, urea, and cholesterol. Exploiting enzymes for the selective detection of target molecules are very good but may not be easily accomplished as enzymes often need to be immobilized onto support materials, which requests either modifying the surface or using appropriate linkers to synthesize biosensors. Currently, many biosensors' signal strength and stability still are low, which encourages the fabrication of functionalized nanomaterials/nanoparticles to enhance electrochemical performance. The review covers current progress in modifying enzyme immobilized biosensors to detect biologically essential molecules (glucose, urea, cholesterol). A discussion of the different types of enzyme immobilization is also provided. This review may be helpful for researchers to build rational and novel biosensors for efficient enzyme immobilization and sensing applications.