Bioprocesses based on surface-associated microorganisms are emerging in environmental and industrial areas due to the physiological specificities and heterogeneities of biofilm cells. This study describes a simple and accurate method for evaluating the recombinant protein expression at a single-cell scale during Escherichia coli biofilm development. The model recombinant protein used was enhanced the green fluorescent protein (eGFP), as its intrinsic fluorescence allows us to quantify expression at both population and single-cell levels. The specific cell fluorescence intensity sharply increased during the first 4days of biofilm cultivation. Thereafter, it decreased abruptly reaching a low-level plateau until the end of the experiment. During biofilm development, the population became increasingly heterogeneous with regard to eGFP expression. Three distinct biofilm types were observed over the course of the experiment: one with a homogeneous population (days 3-5), the second with a moderately heterogeneous population (days 6-8) and the third with a strongly heterogeneous population (days 9-11). Observation of E. coli biofilms by confocal laser scanning microscopy revealed marked spatial heterogeneity, with the cells actively producing eGFP restricted to the top layer of the biofilm. The proposed methodology allows a fine analysis of the recombinant protein expression within E. coli biofilms, and it may be used to optimize the processing conditions.