Background: Enzyme-based diagnostics remain a cornerstone of laboratory medicine, yet advancements in chemistry and microbiology offer new opportunities to optimize their sensitivity, specificity, and application. This study explores the integration of enzymology, microbial profiling, and clinical diagnostics to improve enzyme-driven detection methods. Objective: To experimentally develop, optimize, and validate enzyme-based diagnostic assays by linking chemical substrate modification, microbiological enzyme activity detection, and clinical biomarker evaluation. Methodology: An experimental study was conducted in which HRP, ALP, and β-galactosidase enzymes were chemically optimized and tested using spectrophotometric and fluorometric assays. Nanozyme analogs were also synthesized. Thirty clinical bacterial isolates were evaluated using enzyme activity tests and compared with CRISPR-Cas13 assays. Fifty clinical blood/serum samples were analyzed for ALT, AST, and CRP levels using in-house developed enzyme-based kits, and results were validated against automated laboratory systems. Results: Enzyme assays showed strong catalytic efficiency (e.g., Km = 0.23 mM for HRP-TMB). Microbial identification achieved 93.3% sensitivity and 100% specificity, outperforming some molecular methods. Clinical validation demonstrated high correlation (r = 0.91, p < 0.001) with standard lab results, and ROC analysis showed AUC values above 0.91 for all biomarkers. Nanozymes exhibited enhanced thermal stability. Conclusion: Cross-disciplinary enzyme-based diagnostics are effective, low-cost, and scalable for clinical and microbiological applications. The integration of chemical, microbial, and clinical methods results in robust diagnostic tools suitable for both advanced laboratories and low-resource settings. Future developments should focus on digital integration and multiplexing for broader healthcare impact.