Pyocyanin is a virulence factor produced as a secondary metabolite by the opportunistic human pathogen, Pseudomonas aeruginosa. Fast and direct detection of pyocyanin is of importance as it could provide important insights regarding P. aeruginosa's virulence mechanisms. Here, we present an electrochemical sensing platform of redox-active pyocyanin using transparent carbon ultramicroelectrode arrays (T-CUAs), which were made using a previously reported simple fabrication process (Duay et al. Anal. Chem. 2015, 87, 10109). Square-wave voltammetry was used to quantify pyocyanin concentrations on T-CUAs with and without chitosan gold nanoparticles (CS/GNP) and planar transparent macroelectrodes (T-Macro). The response time (RT), limit of detection (LOD), and linear dynamic range (LDR) differ for each electrode type due to subtle influences in how the detectable signal varies in relation to the charging time and resistive and capacitive noise. In general lower LODs can be achieved at the consequence of smaller LDRs. The LOD for T-Macro was 0.75 ± 0.09 μM with a LDR of 0.75-25 μM, and the LOD for the CS/GNP 1.54 T-CUA was determined to be 1.6 ± 0.2 μM with a LDR of 1-100 μM, respectively. The LOD for the 1.54T-CUA with a larger LDR of 1-250 μM was 1.0 ± 0.3 μM. These LODs and LDRs fall within the range of PYO concentrations for a variety of in vitro and in vivo cellular environments and offer promise of the application of T-CUAs for the quantitative study of biotoxins, quorum sensing, and pathogenesis. Finally, we demonstrate the successful use of T-CUAs for the electrochemical detection of pyocyanin secreted from P. aeruginosa strains while optically imaging the cells. The secreted pyocyanin levels from two bacterial strains, PA11 and PA14, were measured to have concentrations of 45 ± 5 and 3 ± 2 μM, respectively.