The fluorescence detection of ions and pharmaceutical effluents by using organic chemosensors is a valuable surrogate to the currently existing expensive analytical methods. In this regard, the design of multi-functional chemosensors to recognize desirable guests is of utmost importance. In this study, we first show that levofloxacin (LVO) is able to use as a fluorescent chemosensor for the detection of biologically important Cu 2+ (turn-off) and Fe 3+ (turn-on) ions via independent signal outputs in 100% aqueous buffer solutions. Next, using the reciprocal recognition of LVO and Fe 3+ provides a unique emission pattern for the detection of LVO. This approach exhibited a high specificity to LVO among various pharmaceutical samples, namely acetaminophen (AC), azithromycin (AZ), gemifloxacin (GEM) and ciprofloxacin (CIP) and also showed great antiinterference property in urine. The attractive features of this sensing system are availability, easy-to-use, high sensitivity (limit of detection = 18 nM for Cu 2+ , 22 nM for Fe 3+ and 0.12 nM for LVO), rapid response (5 s) with an excellent selectivity.