Aflatoxin B1 (AFB1) is the most toxic mycotoxin, causing harmful effects on human and animal health, and the rapid and sensitive detection of AFB1 is highly demanded. We developed a simple electrochemical aptasensor achieving rapid detection of aflatoxin B1 (AFB1). A short anti-AFB1 aptamer having a methylene blue (MB) redox tag at the 3'-end was immobilized on the surface of a gold electrode. In the absence of AFB1, a complementary DNA (cDNA) strand hybridized with the MB-labeled aptamer, causing MB apart from the electrode surface and low current of MB. In the presence of AFB1, AFB1 competed with the cDNA in the binding to the MB-labeled aptamer, and the aptamer-AFB1 binding caused formation of a hairpin structure, making the MB close to the electrode surface and current of MB increase. Under optimized conditions, we achieved detection of AFB1 over dynamic concentration range of 2 nM-4 μM by using this signal-on electrochemical aptasensor. This method only required a simple 5-min incubation of sample solution prior to rapid electrochemical sensing, more rapid than other electrochemical aptasensors. The sensor could be well regenerated and reused. This sensor allowed to detect AFB1 spiked in 20-fold diluted beer and 50-fold diluted white wine, respectively. It shows potential for detection of AFB1 in wide applications.