Dissolved oxygen (DO) is an important parameter to monitor in processes applicable in, for example, water technology and fermentation. In this study, we report the manufacturing of a membrane-less amperometric sensor that is based on the electrocatalytic reduction of oxygen. The sensor was tested in pH-neutral KNO3 solutions and in a microbial fermentation to monitor the consumption of dissolved oxygen. The nitrogen and boron-doped reduced graphene oxide (N,B-HRGO) is used as an electrocatalyst for oxygen reduction. One step co-doping of nitrogen and boron on graphene oxide is performed using the hydrothermal method. The sensor responded linearly to the DO concentration. A sensitivity of 0.2 mu A/mg.L-1 O-2 is obtained for the DO concentration of 1.5 to 10 mg.L-1 O-2. The membrane-less N,B-HRGO based DO sensor is successfully tested in an Amycolotopsis methanolica fermentation by monitoring the dissolved oxygen in real-time. The sensor detected the consumption of oxygen during the growth of A. methanolica, which shows the compatibility of N,B-HRGO as electrode material for amperometric measurement of dissolved oxygen in aerobic fermentation. This membrane-less amperometric sensor can be used to fabricate microdevices for microbioreactor applications.