Combination of aptamers with DNAzymes attracted intense attention for development of DNA-based biosensors for detection of mycotoxins. In the present study a combination of aflatoxin B1 specific aptamer and HRP- (horseradish peroxidase-) mimicking DNAzyme was optimized for detecting aflatoxin B1. Detecting approach is based on the binding affinity of aflatoxin B1 to its specific aptamer and conversion of substrate to a detectable colorimetric signal by a linked DNAzyme. Compared to conventional methods for aflatoxin B1 detection, DNA-based assay has the advantages of low cost, long-term stability, and rapid, simple, and user-friendly steps.