A label-free, photoluminescence (PL) based biosensor for 2,4,6-trinitrotoluene (TNT) detection was developed by functionalizing diatom biosiilica with a TNT-specific single-chain variable fragment (scFv). The scFv loading was estimated to be 0.040±0.003 (μg scFv/μg biosilica). In saturated concentration, TNT binding to scFvbiosilica quenched about 13% of its PL. Dose response follows Langmuir isotherm with half saturated concentration at 3.66×10⁻⁸ M. The method detection limit (MDL) was estimated to be 3.87×10⁻⁸ M. No interference was observed in the presence of non-complementary explosives. Limitations like small signal intensity and high background signal can be address by optimizing combination of transducer and bioreceptor. In addition, the biosensor system has the potential to be self-assembled by genetically modified diatom, eliminating the whole chemical functionalization steps. This biosensor shows potential for low-cost, high-sensitivity, on-site and selective TNT detection.