The excessive use of antibiotic ofloxacin (Oflx) can cause serious detrimental effects to human health. Therefore, the utmost research priority is required to develop facile methods to detect Oflx. Herein, a V‐shaped aggregation‐induced emission (AIE) active Schiff base SDANA was introduced for the fluorescent turn‐on detection of Oflx. The Schiff base SDANA was synthesized by condensing 4,4'‐sulfonyldianiline with two equivalents of 2‐hydroxy‐1‐naphthaldehyde. The nearly non‐fluorescent SDANA in DMSO showed strong orange emission with the increase in HEPES buffer (H2O, 10 mM, pH 7.4) fractions in DMSO from 70% to 95% due to the combined effects of AIE and ESIPT. The DLS and SEM analyses were performed to complement the formation of self‐aggregates of SDANA. With the addition of Oflx, the fluorescence emission of AIE luminogen (AIEgen) SDANA (lem = 575 nm, lex = 400 nm) was blue‐shifted and enhanced at 530 nm. The interactions of Oflx over the surface of SDANA aggregates disrupted the intramolecular charge transfer and aggregation morphology of SDANA, which gave a distinct fluorescence response to detect Oflx. The detection limit for Oflx was estimated as 0.81 µM, and the developed probe AIEgen SDANA was applied for the quantification of Oflx in human blood serum.