Based on graphite-like carbon nitride (g-CN) nanocomposites coupled with aptamer, a regenerable electrochemiluminescence (ECL) biosensor is developed for the quantitative detection of aflatoxin B ₁ (AFB ₁). In the existence of AFB ₁, the structure of the aptamer changed into a loop, and the original ECL intensity was reduced owing to the enhancement of luminescence quenching between the ferrocene modified at the end of the aptamer and the luminescent substrate g-CN. Moreover, AFB ₁ with oxidation state could also react with high energy state g-CN, leading to further reduction of the electrochemiluminescence signal. At optimum conditions, ECL intensity was decreased in linearity with an AFB ₁ concentration range from 0.005 ng/mL to 10 ng/mL, and the minimum detectable concentration was down to 0.005 ng/mL, which realized trace detection demand with high sensitivity. It was selective for AFB ₁ and its performance had been verified on rice samples, which indicated a promising applying prospect of non-enzymatic electrochemiluminescence AFB ₁ detection.