In this study, we designed a picolinic acid-functionalized calix[4]pyrrole ligand (PCACP). When testing out the fluorescence study with selected antibiotics, we observed remarkable enhancement of fluorescence spectra in presence of ciprofloxacin, singling out the PCACP_Ciprofloxacin complex. This complexation phenomenon was further explored via NMR and FTIR complexation studies to determine the exact binding mechanism of ciprofloxacin discrimination. Further to confirm the experimental study, detailed computational investigations were conducted including the NBO, Mulliken charge and bond length analysis. The results of experimental and computational data provided the contributing role of hydrogen bonding and π- π stacking, favouring the ciprofloxacin discrimination in compared to other tested antibiotics. The limit of detection was calculated to be 18µM, and the developed PCACP ligand showed excellent specificity and could be used to detect ciprofloxacin in water samples. The outcome of this research provides new, effortless fluorescence approach to monitor the presence of ciprofloxacin in water.