An ultrasensitive and rapid detection of trace antibiotics is imperative for food safety and public health. Herein, we present a ratiometric fluorescent sensing strategy based on an aptamer labeled with a fluorescent dye and a highly stable zirconium-porphyrin MOF (PCN-222) as a fluorescence quencher for the high-efficiency detection of chloramphenicol (CAP). PCN-222 exhibits a strong adsorption ability toward the dye-labeled aptamer through π-π stacking, electrostatic, hydrogen bond, and coordination interactions. Experimental and simulation studies confirm that PCN-222 demonstrates a high quenching efficiency via fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) processes. In the presence of CAP, dye-labeled aptamers are released from the PCN-222 surface, resulting in the recovery of fluorescence. This proposed biosensor allows the complete detection of CAP within 26 min. For ratiometric measurement, its detection limit is as low as 0.08 pg mL-1 with a wide detection range from 0.1 pg mL-1 to 10 ng mL-1. It is successfully applied to analyze CAP in milk and shrimp samples, and its results are consistent with those of the commercial ELISA kit. This biosensor not only enables the rapid, ultrasensitive, and highly specific detection of CAP but also reveals excellent universality and multiplexed analysis performance.