A new protein detector of 2,4,6-trinitrophenol (TNP, picric acid) is concisely reported herein for the first time. In brief, the gene of a specific scFv fragment, namely 3.5, was fused separately with the gene of β-lactamase and, subsequently, expressed in the periplasmic space of Escherichia coli, affording in such a way the recombinant fusion protein 3.5-scFv-β-lactamase. This bifunctional immunoreagent tool was further convincingly demonstrated for being capable to directly detect trinitrophenol-tris(hydroxymethyl)aminomethane (TNP-Tris), a less toxic TNP derivative of choice compared to TNP itself, with competitive sensitivity (50 ± 2 fmol or 175 ± 6 pg/mL).