The development of non-toxic, cost-effective and high fluorescent sensing materials has earned significant interest in the last decade. In this work, a simple synthesis technique of mesoporous hydrogen-bonded organic frameworks (HOFs) suitable for the ultrasensitive detection of a commonly used antibiotic, enrofloxacin (ENR), has been reported. The fluorescence of the HOF is completely quenched after formation of a HOF-Cu²⁺ complex as a turn off sensor which undergoes a turn-on mechanism in presence of ENR. The competitive binding of ENR displaces Cu²⁺ ions, thereby restoring the fluorescence of the free HOF. The quenching effect of the HOF's fluorescence in the presence of Cu 2+ ions was quantitatively analysed, establishing a limit of detection (LOD) of 57 nM. The formation of a Cu²⁺-ENR conjugate upon ENR addition leads to the recovery of fluorescence intensity. The mechanism of competitive binding was validated by kinetic studies and density functional theory (DFT) calculations. The sensor demonstrated a linear response to ENR concentrations ranging from 0.01 to 50 μM, with LOD of 70 nM. The high sensitivity with excellent selectivity towards the antibiotics in presence of other interferences highlight the potential for real-time biological applications of this sensing platform.