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A novel microelectrode array sensor was fabricated using MEMS technology on a needle, and then applied to real-time measurement of dissolved oxygen (DO) inside biofilms. The sensor consists of eleven gold disk microelectrodes, a rectangular auxiliary electrode along them, and an external and internal reference electrode. Three kinds of sensors were designed and their responses were characterized and evaluated under various conditions. The arrays exhibit a linear response to DO in the 0–8 mg·L⁻¹ concentration range in water, high sensitivity, repeatability, and low limits of detection (<0.11 mg·L⁻¹ of DO) and quantification (0.38 mg·L⁻¹ DO). The sensors were validated against a commercial Clark-type microelectrode and applied to profiling of DO in a heterotrophic biofilm cultivated in a flat-plate bioreactor. It is shown that the sensor array can provide a multipoint, simultaneous snapshot profile of …