Biodegradation process modeling is an essential tool for the optimization of biotechnologies related to gaseous pollutant treatment. In these technologies, the predominant role of biofilm, particularly under conditions of no mass transfer limitations, results in a need to determine what processes are occurring within the same. By measuring the interior of the biofilms, an increased knowledge of mass transport and biodegradation processes may be attained. This information is useful in order to develop more reliable models that take biofilm heterogeneity into account. In this study, a new methodology, based on a novel dissolved oxygen (DO) and mass transport microelectronic array (MEA) sensor, is presented in order to characterize a biofilm. Utilizing the MEA sensor, designed to obtain DO and diffusivity profiles with a single measurement, it was possible to obtain distributions of oxygen diffusivity and biokinetic parameters along a biofilm grown in a flat plate bioreactor (FPB). The results obtained for oxygen diffusivity, estimated from oxygenation profiles and direct measurements, revealed that changes in its distribution were reduced when increasing the liquid flow rate. It was also possible to observe the effect of biofilm heterogeneity through biokinetic parameters, estimated using the DO profiles. Biokinetic parameters, including maximum specific growth rate, the Monod half-saturation coefficient of oxygen, and the maintenance coefficient for oxygen which showed a marked variation across the biofilm, suggest that a tool that considers the heterogeneity of biofilms is essential for the optimization of biotechnologies.