The design of suitable fluorescent chemosensors for the trace detection of emerging organic pollutants attracted great research interest because of their serious menace to the environment and health hazards. Herein, we report two 1,8-naphthalimide-based fluorescent chemosensors, N-phenyl-4-amino-1,8-naphthalimide (Nap-1) and N-phenyl-3-amino-1,8-naphthalimide (Nap-2), for the discriminative fluorescent sensing of nitro-containing antibiotics. Nap-1 and Nap-2 have been synthesized in two steps from their respective nitro-1,8-naphthalic anhydride precursors and they were fully characterized using standard spectroscopic techniques such as FT-IR, and NMR (1H, and 13C), and HRMS. Both Nap-1 and Nap-2 are highly emissive due to internal-charge transfer (ICT) transition and exhibited a positive solvatochromism in different solvent mediums. The fluorescence titration studies of Nap-1 and Nap-2 with various antibiotics showed high selectivity and strong binding affinity for nitro-containing antibiotics with a parts-per-billions (ppb) level of sensitivity. The initial fluorescence emission intensity of Nap-1 and Nap-2 was quenched upon the addition of nitro-antibiotics such as Nitrofurazone (NFZ) and Dimetridazole (DMZ). Notably, the co-existence of other competing antibiotics showed negligible to moderate fluorescence quenching. The observed discriminative fluorescence sensing was also reflected by sharp visual colour changes. The Stern-Volmer quenching constants (KSV) were calculated to be in the order of 104 M−1. These studies suggest that Nap-1 and Nap-2 can be used as potential “turn-off” fluorescent chemosensors for the selective, sensitive, and fast detection of nitro-antibiotics. © 2022 The Author(s)