Chloramphenicol (CAP) is widely used in treating bacteria infection in animals and humans. However, the accumulation of CAP in food and environment caused serious health risk to human. Consequently, sensitive and selective detection of CAP is of great importance in environmental monitoring and food safety. Among various analytical methods, aptamer-based biosensors exhibit great potentials for CAP detection. Here, we developed an aptamer-based biosensor for rapid fluorescence detection of CAP based on pyrene excimer switch by using a newly selected short DNA aptamer with high affinity. The aptamer was labeled with pyrene molecules at both ends. The binding of CAP to the aptamer probe caused two pyrene molecules close to each other and the formation of a pyrene excimer, which induced the increase of the fluorescence signal from the pyrene excimer. CAP detection was achieved by measuring the fluorescence signal changes of the aptamer probes with dual pyrene labels. Under optimized conditions, the developed aptamer biosensor showed a detection limit of 24.4 nmol/L for CAP. The aptamer-based fluorescence sensor could quantify CAP in diluted tap water and lake water, exhibiting potentials for the application in real sample sensing of CAP.