Determination of aflatoxin B1 (AFB1) is still a big issue in food safety. In this paper, we developed a luminescence AFB1 detection method combined with ATP-releasing nucleotides (ARNs) and AFB1 aptamer. Firstly, using a new coupling method, we synthesized two ARNs (dTP₄A and dGP₄A) in a yield of 67% and 58%, respectively. The newly prepared ARNs show a much lower background. Then, we developed a new isothermal polymerase amplification method. In this method, two DNA hairpins were used to substitute the circle DNA template in rolling circle amplification. Using this amplification method and combined with AFB1 aptamer, a new AFB1 detection method is developed. A detection limit as low as 0.3 pM is achieved. This method is simple and efficient, and will have a great potential to be used for food safety and public health.