NIR-emissive ESIPT active PBI-keto/enol assemblies have been developed for the detection of 2, 6-dichloro-4-nitroaniline (DCN). These assemblies show ‘on-on’ optical response towards DCN due to combined ESIPT-AIEE phenomenon with a detection limit of 1.65 nM. The potential of PBI-keto/enol assemblies to detect DCN has also been explored in grapes juice/grape residue, and soil for six consecutive days. Further, the biological applications of PBI-keto/enol assemblies to detect DCN in blood serum and to image DCN in live cells and to restrict the DCN induced cell death has been demonstrated in MG-63 cell lines.