An on-chip whole-cell bioassay has been carried out using Escherichia coli tester strains for genotoxicity. In this assay format, the mutagen-responsive bioluminescence (BL) strains are immobilized in a chip assembly in which a silicon chip is placed between two poly(dimethylsiloxane) (PDMS) chips. In the chip assembly, microchannels fabricated on the two separate PDMS layers are connected via perforated microwells on the Si chip, and thus a three-dimensional microfluidic network is constructed. The strains mixed with agarose are loaded from the channels on one of the two PDMS layers into the wells on Si chip, followed by gelation. Induction of the expression of firefly luciferase in the tester strains and BL reaction are successively carried out by filling the channels on another PDMS layer with samples containing inducer (genotoxic substance) and then adenosine triphosphate/luciferin mixture, respectively. BL emission from each of the wells can be monitored by using a charge-coupled device camera to obtain an overall picture of the chip. The on-chip format based on a three-dimensional microfluidic network provides a combinatorial bioassay for multiple samples with multiple tester strains in a simple chip assembly. Thus, the presented method could be applied not only to various microbial sensing applications but also to other (bio)chemical analyses.