Herein, an aggregation-induced emission (AIE) luminogen (AIEgen) NANH was developed by condensing equimolar amounts of 2-hydroxy-1-naphthaldehyde and 3-hydroxy-2-naphthohydrazide. The AIE behaviour of NANH was explored in a mixed DMSO-H2O (HEPES buffer, pH 7.4, 10 mM) medium. The self-aggregates of NANH show enhanced emission at HEPES fractions (fHEPES) ≥70%. AIEgen NANH (fHEPES 95%, λex = 390 nm, λem = 490 nm) was applied for the fluorescence turn-off sensing of sparfloxacin and azithromycin. With the addition of different analytes, the fluorescence intensity of AIEgen NANH was quenched by sparfloxacin and azithromycin. No noticeable changes in the fluorescence of AIEgen NANH were observed with other added analytes. Experimental evidence supported that the fluorescence quenching of AIEgen NANH by sparfloxacin and azithromycin is static in nature. From the fluorescence titrations, the detection limits of AIEgen NANH were estimated down to 0.64 μM and 0.65 μM for sparfloxacin and azithromycin, respectively. Further, blood serum samples spiked with sparfloxacin and azithromycin were used to examine the analytical utility of AIEgen NANH.