Contamination of feed and food by aflatoxin B1 (AFB1), one of the most toxic of the mycotoxins, is a global concern. To prevent food safety scares, and avoid subsequent economic losses due to the recall of contaminated items, methods for the rapid, sensitive and specific detection of AFB1 at trace levels are much in demand. In this work, a simple, ultrasensitive, and reliable aptasensor is described for the detection of AFB1. An AFB1 aptamer was used as a molecular recognition probe, while its complementary DNA played a role as a signal generator for amplification by real-time quantitative polymerase chain reaction (PCR). Under optimal conditions, a wide linear detection range (5.0×10−5 to 5.0ngmL−1) was achieved, with a high sensitivity (limit of detection (LOD)=25fgmL−1). In addition, the proposed aptasensor exhibited excellent specificity for AFB1 compared with eight other mycotoxins, with no obvious Ct value change. This aptasensor can also be used in quantifying AFB1 levels in Chinese wildrye hay samples and infant rice cereal samples, demonstrating satisfactory recoveries in the range of 88–127% and 94–119%, respectively. This detection technique has a significant potential for high-throughput, quantitative determination of mycotoxin levels in a large range of feeds and foods. •We developed a novel aptasensor based on RT-qPCR for detection of AFB1.•This aptasensor is the most sensitive for AFB1 up to now with a limit of detection of 25fgmL−1.•The selectivity of this aptasensor is excellent for AFB1 even in the presence of its analogues such as AFB2 and AFM1.•This aptasensor could be applied to high-throughput quantification of AFB1 at trace levels in feeds and foods.