This thesis pioneers two novel and independent strategies in catalytic biosensors designed to address their critical challenges in stability, cost, and practicality for mastitis infection and early-stage cancer diagnosis. Mastitis infection is a major cause of economic loss in the dairy industry and its diagnosis can help reduce financial losses and improve overall herd health. The first approach employs an enzyme cascade system for electrochemical lactose detection in dairy products. By integrating β-galactosidase and glucose oxidase enzymes, lactose is hydrolyzed into glucose, which is further oxidized to generate electroactive H2O2. A RuO2-multiwalled carbon nanotube nanocomposite mediates the electrochemical oxidation reaction of H2O2 and achieving a detection limit of 0.036 mM with high reproducibility (RSD < 3%) in phosphate buffer, with a recovery rate high of 99.6% in milk sample. Validated in milk samples, this system demonstrates industrial adaptability via flow injection analysis, offering a robust solution for food quality and mastitis infection diagnosis. However, limitations in enzyme stability under harsh conditions and complex purification processes prompted the exploration of nanozymes (NZs) as superior alternatives. Cancer is the second leading cause of death, and an early-stage diagnosis can significantly decrease the mortality rate since medication is more efficient at early-stage. The second study presents tyrosine-capped platinum nanozymes (Pt-Tyr NZs), synthesized through chemical reduction, which mimic peroxidase-like activity. These NZs are conjugated with capture DNA (cDNA), effectively suppressing their catalytic activity. Upon hybridization with the cancer biomarker miRNA-21, the cDNA detaches, restoring the nanozymes' catalytic function. This mechanism of reversible passivation of catalytic activity of Pt-Tyr NZs enables rapid, one-step spectrophotometric detection of miRNA-21 with a limit of detection of 10.8 nM with high reproducibility (RSD < 1%, n = 4) and repeatability (RSD < 1%, n = 4) in saline phosphate buffer, and 109.7% recovery percentage for miRNA-21 measurement spiked in serum sample and minimal interference. Compared to natural enzymes, NZs exhibit superior stability across pH/temperature variations, cost-effective synthesis, and tunable activity through surface modification like cDNA conjugation. Their simplicity in preparation and adaptability to point of care (POC) detection shows their potential in clinical analysis for early-stage cancer diagnosis. This highlights NZs’ ability to overcome traditional enzymatic limitations while delivering scalable, precise, and durable solutions for application in real sample analysis. The presented enzyme cascades and NZ-cDNA systems, in this work advances catalytic biosensing through parallel innovations where enzyme-based platforms can be used in food industries and NZ-cDNA conjugate in clinical diagnostics.