Enzyme-based biosensors have emerged as a transformative technology, leveraging the specificity and catalytic efficiency of enzymes across various domains, including medical diagnostics, environmental monitoring, food safety, and industrial processes. These biosensors integrate biological recognition elements with advanced transduction mechanisms to provide highly sensitive, selective, and portable solutions for real-time analysis. This review explores the key components, detection mechanisms, and applications of and future trends in enzyme-based biosensors. Artificial enzymes, such as nanozymes, play a crucial role in enhancing enzyme-based biosensors by mimicking natural enzyme activity while offering improved stability, cost-effectiveness, and scalability. Their integration can significantly boost sensors' performance by increasing their catalytic efficiency and durability. The functionality of enzyme-based biosensors is built on three essential components: enzymes as biocatalysts, transducers, and immobilization techniques. Enzymes serve as the biological recognition elements, catalyzing specific reactions with target molecules to produce detectable signals. Transducers, including electrochemical, optical, thermal, and mass-sensitive types, convert these biochemical reactions into measurable outputs. Effective immobilization strategies, such as physical adsorption, covalent bonding, and entrapment, enhance the enzymes' stability and reusability, enabling their consistent performance. In medical diagnostics, they are widely used for glucose monitoring, cholesterol detection, and biomarker identification. Environmental monitoring benefits from these biosensors in detecting pollutants like pesticides, heavy metals, and nerve agents. The food industry employs them for quality control and contamination monitoring. Their advantages include high sensitivity, rapid response times, cost-effectiveness, and adaptability to field applications. Enzyme-based biosensors face challenges such as enzyme instability, interference from biological matrices, and limited operational lifespans. Addressing these issues involves innovations like the use of synthetic enzymes, advanced immobilization techniques, and the integration of nanomaterials such as graphene and carbon nanotubes. These advancements enhance enzymes' stability, improve their sensitivity, and reduce the detection limits, making this technology more robust and scalable.