Fluoroquinolone is one of the most widely used class of broad-spectrum antibiotics in the world. Due to its widespread use and misuse, antimicrobial resistance against fluoroquinolones is becoming a matter of global health concern. Antibiotic residues are found in the surface and groundwater through the release of animal and human excretion and pharmaceutical effluents. Hence a robust and cost-effective way to detect antibiotics in relevant matrices is the current need of the hour. We have optimized an inexpensive chemosensing assay that can detect the presence of fluoroquinolone with a limit of detection (LOD) from 1.18 to 2.21 ppb (3.5–6.6 nM) in various environmental matrices and urine as a source of biological source of contamination. The assay has proved reliable against a comprehensive range of interferents. The linear range of operation was obtained from 10 ppb to 1000 ppb with R2 = 0.987–0.996. The recovery result fell between 95% and 104%, demonstrating reasonable accuracy. The assay has also been converted to a filter paper-based device with hydrophobic boundary generated by office printer toner ink to test for counterfeit medicines. The paper chemosensor produces naked eye detectable yellow color instantly in the presence of the analyte. Counterfeit drug testing has been reported against a broad range of over-the-counter available drugs with nearly no sample preparation. The paper sensor was found to be stable and functional over a year after storing it at room temperature.