Understanding of the processes taking place inside biofilms is a key parameter to progress in the optimization of biofiltration technologies. This study was conducted with the aim of developing a novel dissolved oxygen (DO) microsensor specially designed for biofilms monitoring. The microsensor was fabricated through standard photolithography techniques, resulting in a microelectrodes array (MEA) of 11 gold circular working electrodes, with a diameter of 50 µm , and a gold reference electrode, which allows obtaining a snapshot oxygen profile of 1 mm of depth. The performance of the sensor was fully characterized under different conditions, inwhich the sensor presented high sensitivity and repeatability, and low detection and quantification limits. Monitoring of sensor performance showed a stable and reliable response. The developed sensor was used in obtaining micropofiles in an aerobic heterotrophic biofilm, showing similar response to Clark-type commercial microsensors. These studies concluded that the novel MEA sensor for DO monitoring allows obtaining oxygen profiles within biofilms, becoming a useful tool for the research of many biological applications.