Pyocyanin, a toxin produced by Pseudomonas aeruginosa, offers potential as a biomarker for the indirect detection of this bacterium of major importance for infections in burns, woundcare and cystic fibrosis. Pad-printed carbon electrodes are herein explored using square wave voltammetry to detect pyocyanin in a range of buffered and biological media. Third-order polynomial baseline fitting was explored to enhance the analytical sensitivity and extend the linear range to submicromolar concentrations. These modelling baselines showed excellent correlation with the experimental data, confirmed by high Interclass Correlation Coefficients of 0.995–0.998, and enabled the quantification of pyocyanin – with linearity extended down to 0.18 μM in Human Serum and 0.336 μM in both Britton-Robinson buffer and Simulated Wound Fluid, and derived Limits of Detection of 0.17, 0.15 and 0.09 μM, respectively, in this proof-of-concept study. Therefore, the use of very simple, cost-effective printed carbon materials enabled the detection of clinically relevant concentrations of this important biomarker through a new baseline fitting model and offers a novel approach for point-of-care diagnostics where Pseudomonas aeruginosa infections are critical.