The fluoroquinolone antibacterial agent fleroxacin (FLO) has been widely used for the treatment of urinary infections. Quantitatively analyzing the amount of FLO is vital for human health and the ecological system, but still proves challenging due to the complex environmental interference. In this work a ratiometric fluorescence sensor MOF-808-TPY is successfully constructed by incorporating a functional ligand, 2,2′:6′,2′'-terpyridine-4′-carboxylic acid (TPY), to the zirconium node of MOF-808 via post-synthetic modification. With the addition of fleroxacin, the prepared MOF-808-TPY sensor exhibits decreased fluorescence intensity at 370 nm, while the intrinsic fluorescence from fleroxacin at 440 nm is greatly enhanced. The yielded ratiometric response can effectively eliminate interference from environmental factors, and is able to discriminate FLO from other fluoroquinolone antibiotics efficiently, demonstrating high selectivity towards FLO analysis. A linear relationship of the fluorescence ratio F370/F440 against the concentration of FLO is observed in the range of 0 ∼ 13.50 μM with the detection of limit down to 0.01 μM. Excellent recoveries of FLO levels in real water samples for practical application are also achieved. Additionally, MOF-808-TPY could be easily integrated onto nanofibers for FLO sensing in aspect of potential sensing device. This work represents a promising strategy for constructing MOFs-based ratiometric sensors in monitoring fluorescent antibiotics.