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Pseudomonas aeruginosa is the most prevalent cause of diabetic wound infection and amputation. Pyocyanin is one of the popular markers to identify Pseudomonas infections. However, no existing sensors are on the verge of being used as a suitable sensor for detecting pyocyanin in real-world applications. This study employed a physically cross-linked approach to decorate the surface of a working electrode with a multiwalled carbon nanotube (MWCNTs/PVA) nanocomposite, which increases electron transport, sample absorption, and biocompatibility. Under optimal conditions, the electrochemical behavior of a pyocyanin sensor was investigated using cyclic voltammetry and square wave voltammetry. The developed pyocyanin sensor demonstrated an excellent analytical range covering a clinically relevant range of 1–100 μM pyocyanin in various sample matrices including phosphate buffer, Lysogeny broth, simulated wound exudate, and Mueller–Hinton broth, showing a detection limit of 0.48, 0.10, 1.63, and 0.39 μM, respectively, with R 2 > 0.99. The sensor’s potential wearability and point-of-care capability were demonstrated by detecting pyocyanin in wounds on ex vivo porcine skin and an in vivo animal model with excellent biocompatible property, achieving cell viability of L-929 > 80%. The detection could be performed directly in various sample matrices without any preparation processes. The sensor demonstrated high selectivity and sensitivity in the presence of common wound interferences. As a result, the proposed method has the potential to be a promising sensor for the early detection of P. aeruginosa infection in wounds in the perspective of wound care technology in the future.