Systems designed to sensitively and accurately detect whole pathogen particles, their components, or other proteins diagnostic of infection or disease are of interest as sensors for biodefense and clinical diagnostics. To this end, we examined the potential of a sensing strategy based on live T-cell/B-cell interactions in a biosensor chip format. An approach to fabricate patterned hydrogel microwells functionalized at their bases with antibodies to promote specific immobilization of lymphocytes was developed and used to array single T cells in a regular pattern on a substrate. A sensing platform was created by overlaying arrayed T cells with a confluent layer of antigen-capturing B cells. In this system, a peptide analyte added to the chip was captured by B cells and physically presented to arrayed T cells by B-cell-surface major histocompatibility complex molecules, triggering T cells through their T-cell receptors. T-cell recognition of the target peptide was detected by fluorescence measurements of T-cell intracellular calcium levels, a biochemical read-out of T-cell receptor triggering. We demonstrate that this approach allows rapid, sensitive detection of a model peptide analyte, and that T-cell arrays allow for maximal T-cell/B-cell contacts while simultaneously optimizing single-cell fluorescence detection for analysis of the sensor response. This approach could be of interest for the design of sensing platforms that can detect both peptide fragments and whole intact pathogens, irrespective of surface mutations that might be induced naturally or during “weaponization”.