<p>Resazurin is a redox-active dye widely employed as a metabolic indicator, yet its detailed electronic properties across protonation states and their implications for electrochemical applications remain underexplored. Here, we combined density functional theory (DFT) and electrochemical analyses to investigate the pH-dependent electronic and redox behavior of resazurin and its reduction product, resorufin. DFT calculations revealed that the predominant neutral species of resazurin at pH 7 shares similar HOMO–LUMO energy levels and global reactivity descriptors with resorufin, though resazurin exhibits higher electrophilicity, consistent with its more positive experimental reduction potential. Principal component analysis further demonstrated clustering of resazurin species according to protonation state, highlighting strong shifts in electronic stability under acidic and alkaline conditions. Cyclic voltammetry experiments confirmed these theoretical predictions, showing pH-dependent modulation of peak currents and redox equilibria, with enhanced electron transfer kinetics under alkaline conditions. Importantly, square wave voltammetry (SWV) enabled sensitive quantification of resazurin, displaying a linear correlation between peak area and dye concentration (<i>R</i>² = 0.78). Extending this approach to bacterial suspensions, we observed an inverse linear correlation (<i>R</i>² = 0.895) between turbidity and voltammetric peak area, enabling indirect bacterial quantification via metabolic reduction of resazurin. Taken together, our findings provide the first integrated DFT-electrochemical framework for resazurin, revealing novel insights into its protonation-dependent electronic properties and demonstrating the utility of SWV for both dye quantification and bacterial detection.</p> Graphical abstract <p></p>