New methods for functional assays of chemical receptors are highly essential for the research of chemical signal transduction mechanisms and for the development of chemical biosensors. This study described a novel bioengineered cell-based biosensor for label-free functional assays of chemical receptors by localized extracellular acidification measurement with a light-addressable potentiometric sensor (LAPS). A human taste receptor, hT2R4, and an olfactory receptor of Caenorhabditis elegans (C. elegans), ODR-10, were selected as models of chemical receptors, which were expressed on the plasma membrane of human embryonic kidney (HEK)-293 cells. The specific ligand binding function of expressed chemical receptors was monitored by localized extracellular acidification measurement using LAPS chip with a movable focused laser illuminating on the desired single cell. The function of expressed olfactory receptors was further validated using MDL12330A, which can specifically inhibit the activity of adenylyl cyclase. The obtained results indicate that both of chemical receptors were successfully expressed in HEK-293 cells and can be functionally assayed by this bioengineered cell-based biosensor that shows dose-dependent responses to the target ligands of chemical receptors. This bioengineered cell-based biosensor exhibits the sensitivity of 1.0 mV/s for hT2R4 assays, and 9.8 mV/s for ODR-10 assays. The negative control cells without any chemical receptor expression show no response to all the chemical stimuli tested. All the results demonstrate this bioengineered cell-based biosensor can be used to detect the interactions between chemical receptors and their ligands. This provides a valuable and promising approach for label-free functional assays of chemical receptors as well as for the research of other GPCRs.