A facile, sensitive, and label-free aptamer-based fluorescent biosensor (aptasensor) for chloramphenicol (CAP) detection was successfully developed based on an aggregation-induced emission (AIE) probe and graphene oxide (GO). In this aptasensor, the specific aptamer of CAP (C-Apt) is used as the recognition part, an AIE molecule, 9,10-distyrylanthracene (DSA) derivative with short alkyl chains (9,10-bis{4-[2-(N,N,N-trimethylammonium)-ethoxy]styrene}anthracene dibromide, DSAC2N), as the fluorescent probe, and GO with a low oxidation degree as the fluorescent quencher. Initially, the AIE probe DSAC2N and C-Apt could be adsorbed on GO through π-stacking interactions, and the fluorescence of DSAC2N could be efficiently quenched due to the energy transfer between DSAC2N and GO. When CAP is added, C-Apt can preferentially bind with CAP and the newly formed complex (C-Apt-CAP) can be released from GO, resulting in the recovery of the fluorescence signal of DSAC2N. Thus, with the aid of GO, turn-on detection of CAP can be readily realized by monitoring the fluorescence signal of DSAC2N from "off" to "on". Under the optimized conditions, the aptasensor exhibits a high sensitivity toward CAP with a limit of detection of 1.26 pg/mL. Besides, we have successfully applied this aptasensor to the detection of CAP in spiked milk.