A supramolecular microfluidic optical chemosensor (muFOC) has been fabricated. A serpentine channel has been patterned with a sol-gel film that incorporates a cyclodextrin supramolecule modified with a Tb3+ macrocycle. Bright emission from the Tb3+ ion is observed upon exposure of the muFOC to biphenyl in aqueous solution. The signal transduction mechanism was elucidated by undertaking steady-state and time-resolved spectroscopic measurements directly on the optical chemosensor patterned within the microfluidic network. The presence of biphenyl in the cyclodextrin receptor site triggers Tb3+ emission by an absorption-energy transfer-emission process. These results demonstrate that the intricate signal transduction mechanisms of supramolecular optical chemosensors are successfully preserved in microfluidic environments.