Two bis-heteroleptic ruthenium(II) complexes, 1[PF₆]₂ and 2[PF₆]₂ of 4,7-dihydroxy-1,10-phenanthroline, have been developed for the highly selective ‘light-up’ detection of nerve agent simulant and organophosphate pesticide in aqueous solution. Both the complexes, 1[PF₆]₂ and 2[PF₆]₂, were fully characterized by spectroscopic means and single-crystal X-ray diffraction studies. Compounds 1[PF₆]₂ and 2[PF₆]₂ show a selective turn-on luminescence response in the presence of nerve agent stimulant DCP over other competitive nerve agent surrogates, acylating agents, HCl and pulmonary agent like phosgene. The detection of DCP was achieved via a nucleophilic attack of two hydroxyl groups of the 4,7-dihydroxy-1,10-phenanthroline ligand with DCP by forming bulkier phosphotriester and corresponding PL enhancement. The detection limit implies that 1[PF₆]₂ and 2[PF₆]₂ are able to detect DCP at the micromolar level. ¹H NMR titration, ³¹P NMR and ESI-MS analysis collectively support the binding mechanism of phenolic hydroxyl groups of the ligand L with the PO center of DCP. Triplet state TD-DFT calculations endorsed that the weak and strong red luminescence of 1 and 1-(DCP)₂ originated from the non-emissive ³MC and emissive ³MLCT states, respectively. Also, probes 1[PF₆]₂ and 2[PF₆]₂ have been used for the highly selective luminescence detection of an organophosphate pesticide, dichlorvos, over the market available other organophosphorus pesticides (paraoxon–ethyl, parathion–ethyl, profenofos, and dimethoate). Furthermore, a test paper strip coated with complex 1 was fabricated to detect DCP in the vapor phase and dichlorvos in solution. Finally, probe 1 was successfully applied in biological studies to visualize DCP as well as dichlorvos in catfish brain tissue.