A novel microelectrode array (DO-MEA) sensor was designed and fabricated using microelectromechanical systems technology on a needle for real time measurement of dissolved oxygen (DO). The sensor consisted of eleven gold disk microelectrodes and a rectangular auxiliary electrode along them. The sensor can also be operated with an integrated reference system. Three different sensor designs were fabricated, and their responses were fully characterized and evaluated. The DO-MEA sensor presented a linear response in the 0-8 mg DO·L-1 concentration range in water, displaying high sensitivity and repeatability. Knowledge of bacterial activity inside biofilms is key to the optimization of applied biotechnologies. The developed sensor was validated against a commercial Clark-type microelectrode overcoming its drawbacks, by profiling a heterotrophic biofilm cultivated in a flat-plate bioreactor. The DO-MEA sensor provided a multipoint, simultaneous dissolved oxygen snapshot profile inside a biofilm with high spatial resolution due to its micrometric dimensions, thus becoming a powerful tool for the research of many similar biological-based processes and applications.