In order to develop rapid detection method for chloramphenicol in food, in this study, a novel chloramphenicol aptasensor was constructed based on fluorescence quenching ability of MnO₂ nanosheet and fluorescence amplification ability of Exo-I digestion. Results showed that under optimal fluorescence detection conditions of 50 nmol/L concentration of aptamer, 0.05 mg/mL concentration of MnO₂ nanosheet, 0.4 U/μL concentration of Exo-I and Exo-I digestion time of 50 minutes, and the fluorescence intensity had a good linear relationship with the concentration of chloramphenicol (R²=0.995). The linear range of detection was 0.1~80.0 nmol/L with the detection limit of 0.08 nmol/L. The constructed detection approach had the advantages of simple operation and high sensitivity, and could realize accurate detection of chloramphenicol in food samples.