A novel electrochemical aptasensor for highly sensitive detection of Chloramphenicol (CAP) in honey was successfully developed based on target-induced strand release (TISR). CAP aptamer was immobilized on electrode and then hybridized with the complementary biotinylated detection probe to form aptamer/DNA duplex. In the presence of CAP, the TISR resulted in the dissociation of biotinylated detection probe from the electrode. Then the binding of streptavidin-alkaline phosphatase (ST-AP) to the remaining biotinylated detection probe led to enzyme-amplified electrochemical signal, which decreased with the increase of CAP concentration. Under optimal conditions, the electrochemical signal was linear with the logarithm of CAP concentrations in the range from 1 nM to 1000 nM with the detection limit of 0.29 nM of CAP. In addition, the designed strategy allowed the direct analysis of real honey samples and the results showed a good agreement with conventional LC-MS/MS method. (C) 2012 Elsevier B.V. All rights reserved.