In this work, using DNA and exonuclease-I (Exo-I) as signal amplification strategy, a novel and facile electrochemical aptasensor was constructed for fumonisin B (FB) detection. The G-rich complementary DNA (cDNA) was immobilized onto the electrode surface. Then, aptamer of FB was hybridized with cDNA to form double-stranded DNA. In the absence of FB, double-stranded DNA and G-rich cDNA on the electrode surface promoted effectively methylene blue (MB) enrichment and amplified the initial electrochemical response. In the presence of FB, the combination of aptamer and FB led to the release of aptamer from the electrode surface and the expose of 3' end of single-stranded cDNA. When Exo-I was added onto the electrode surface, the single-stranded cDNA was degraded in the 3'-5' direction. The decrease of double-stranded DNA and G-rich cDNA resulted in the less access of MB to the electrode surface, which decreased the electrochemical signal. The experimental conditions including incubation time of FB, the amount of Exo-I and incubation time of Exo-I were optimized. Under the optimal conditions, the linear relationship between the change of peak current and the logarithmic concentration of FB was observed in the range of 1.0 × 10-1000 ng mL with a low limit of detection of 0.15 pg mL. The experimental results showed that the prepared aptasensor had acceptable specificity, reproducibility, repeatability and stability. Therefore, this proposed aptasensor has a potential application in the food safety detection.