Miniaturized and integrated components for electrochemical detection in micro- and nano-fluidic devices are of great interest as they directly yield an electrical signal and promise sensitive, label-free, real-time detection. One of the challenges facing electrochemical sensing is the lack of reliable reference electrode options. This paper describes the fabrication and characterization of a microscale palladium hydride reference electrode in a single microfabrication step. The reference electrode was integrated inside of a nanoscale constriction along with a gold working electrode to create a complete electrochemical sensor. After charging the palladium electrode with hydrogen, the device was used to detect pyocyanin concentrations from 1–100 μM, with a 0.597 micromolar detection limit. This is the first time that a palladium hydride reference electrode has been integrated with a microfabricated electrochemical sensor in a nanofluidic setup. The device was then used over the course of 8 days to measure pyocyanin produced by four different Pseudomonas aeruginosa strains in growth media. By utilizing square wave and differential pulse voltammetry, the redox active molecule, pyocyanin, was selectively detected in a complex solution without the use of any electrode surface modification.