Pyocyanin is a virulence factor exclusively secreted as a secondary metabolite by the opportunistic human pathogen Pseudomonas aeruginosa. Fast and direct detection of pyocyanin is of importance as it could provide fundamental insights regarding P. aeruginosa’s virulence mechanisms. Here, we demonstrate an electrochemical-sensing platform that is able to quantitatively determine the bio-synthesis and release of redox-active pyocyanin in real time using transparent carbon ultramicroelectrode arrays (T-CUAs). We quantified pyocyanin concentrations on various types of T-CUA electrodes using square-wave voltammetry to determine limits of detection (LODs) and linear dynamic ranges (LDRs). LODs and LDRs fall within the micromolar range for a variety of in vitro and in vivo cellular environments and offer promise of the application of T-CUAs as sensing devices for the quantitative study of biotoxins, bacterial group behavior phenotypes, and pathogenesis. We also demonstrate successful use of T-CUAs for electrochemical detection of pyocyanin secreted from P. aeruginosa strains while optically imaging the cells through the transparent electrodes. Secreted pyocyanin levels from two virulent bacterial strains, clinical PA11 and wild-type PA14, were measured. Finally, we present real-time electrochemical monitoring of pyocyanin using T-CUAs while optically and fluorescently analyzing aggregate formation of various clinical and laboratory bacterial strains.