Developing a selective colorimetric assay for fluoroquinolones (FQs) that show visible color response is highly desired, which is challenging because FQs show extremely similarity in structure and inherently absorb light in the UV color range. In this work, a selective colorimetric assay for pefloxacin (PEF) was fabricated through integrating two rection systems: the inhibition effects of PEF on the AChE-like activities of Al3+ decorated MOF-808 (MOF-808-Al) and the thiol-response oxidase-mimicking reaction of MnO2 nanosheets. The MnO2 nanosheets, due to their oxidase-mimicking properties, can oxidize the colorless 3, 3', 5, 5'-tetramethylbenzidine (TMB) into its blue-colored oxidized form, TMB+. Assisted by the synergistic action of metal-OH and Lewis acid sites, MOF-808-Al exhibits AChE-like activities. These activities catalyze the decomposition of acetylthiocholine into thiocholine. Thiocholine has a stronger reactivity compared to TMB; it can reduce the MnO2 nanosheets, which in turn prevents the color response. In addition, PEF inhibits the AChE-like activities of MOF-808-Al by blocking its active sites. The quantitative detection of PEF is accomplished by recording the blue color response of the system, both in buffer solutions and in commercial meat samples. This assay demonstrates excellent tolerance and selectivity against various interfering substances, antibiotics, and pesticides.