Cell lines expressing recombinant G-protein coupled receptors (GPCRs) are activated by specific ligands resulting in transient [Ca2+] rises that return to basal levels in 30–60 s. Yellow Cameleon 3.6 (YC3.6) is a genetically encoded calcium indicator which can be co-expressed to monitor these cytosolic [Ca2+] changes in real-time using Förster (Fluorescence) resonance energy transfer (FRET). On this basis, we designed the prototype of a generic microfluidic biosensor of GPCR activation, imaging [Ca2+] changes in recombinant human HEK293 cells, which express a combination of a GPCR (Neurokinin 1) and YC3.6. An internal reference for non-specifically induced [Ca2+] changes were YC3.6 cells without GPCR but expressing a red fluorescent protein (mCherry) for identification. These cell lines were grown as a mixed population in a flow cell with a volume of ~50 µl and a flow cell surface of 170 mm2. Cells were activated by brief exposures to specific and non-specific analytes using an injection valve with a flexible sample volume (tested range 5–100 µl) at a flow speed of 100 µl/min. A flow cell surface of 0.2 mm2 with 50 cells was imaged every 2–4 s to obtain signal kinetics. The lower limit of detection was 30 pM Substance P (SP, 2 pg/50 µl), and reproducible responses to repeated injections every 3 min were obtained at 1 nM SP. This biosensor was designed for ~50 cells for statistical reasons, but at a lower limit of 1 receptor- and 1 reference-cell, specific ligand detection is still feasible.