A novel, highly sensitive and stable voltammetric biosensor was developed for accurate creatinine determination in clinical and pharmaceutical applications. Creatinine serves as a crucial biomarker for kidney function assessment, making its precise quantification essential for early detection of renal disorders and monitoring therapeutic interventions. The innovative electrochemical platform incorporates advanced nanomaterial modifications to enhance detection capabilities and analytical performance metrics. The developed voltammetric creatinine biosensors demonstrated exceptional sensitivity with significantly improved signal-to-noise ratios and rapid response times for efficient creatinine quantification. The sensing mechanism relies on specific enzymatic reactions coupled with electrochemical signal transduction, enabling precise biomarker detection across clinically relevant concentration ranges. The biosensor exhibited excellent operational stability and maintained consistent performance characteristics for extended periods. Storage stability tests revealed reliable functionality for at least 4 weeks when preserved in controlled dry environments at optimal temperatures of 4–6 °C. These storage conditions ensure preservation of enzymatic activity and electrode surface integrity. Continuous operation studies demonstrated remarkable durability during prolonged testing periods. The biosensor maintained reproducible and stable electrochemical responses throughout at least 10 hours of constant operation with 1 × 10⁻³ M creatinine solutions prepared in phosphate buffer medium. This operational robustness indicates excellent potential for real-world clinical applications. The developed microsensors were successfully validated through comprehensive testing with pharmaceutical sample matrices. Voltammetric performance characteristics including sensitivity, selectivity, detection limits, and linear response ranges were systematically investigated and optimized.