A ratiometric luminescence intensity imaging approach is presented, which enables spatial O2 measurements in biofilm reactors with transparent planar O2 optodes. Optodes consist of an O2 sensitive luminescent dye immobilized in a 1–10 µm thick polymeric layer on a transparent carrier, e.g. a glass window. The method is based on sequential imaging of the O2 dependent luminescence intensity, which are subsequently normalized with luminescent intensity images recorded under anoxic conditions. We present 2-dimensional O2 distribution images at the base of a tap water biofilm measured with the new ratiometric method and compare the results with O2 distribution images obtained in the same biofilm reactor with luminescence lifetime imaging. Using conventional digital cameras, such simple normalized luminescence intensity imaging can yield images of 2-dimensional O2 distributions with a high signal-to-noise ratio and spatial resolution comparable or even surpassing those obtained with expensive and complex luminescence lifetime imaging systems. The method can be applied to biofilm growth incubators allowing intermittent experimental shifts to anoxic conditions or in systems, in which the O2 concentration is depleted during incubation.