•Ochratoxin A, a possible human carcinogen from the mycotoxins group, was detected.•A DNAzyme–aptamer conjugate and a blocker were designed as bio-recognition element.•The unhybridized DNAzyme activity was linearly correlated with the OTA concentration.•The achieved LOD (4 nM) was suitable for OTA detection in different food samples.•A double liquid–liquid extraction method was developed to purify OTA from wine.We report a new label-free colorimetric aptasensor based on DNAzyme–aptamer conjugate for rapid and high-throughput detection of Ochratoxin A (OTA, a possible human carcinogen, group 2B) in wine. Two oligonucleotides were designed for this detection. One is N1 for biorecognition, which includes two adjacent sequences: the OTA-specific aptamer sequence and the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The other is a blocking DNA (B2), which is partially complementary to a part of the OTA aptamer and partially complementary to a part of the DNAzyme. The existence of OTA reduces the hybridization between N1 and B2. Thus, the activity of the non-hybridized DNAzyme is linearly correlated with the concentration of OTA up to 30 nM with a limit of detection of 4 nM (3σ). Meanwhile, a double liquid–liquid extraction (LLE) method is accordingly developed to purify OTA from wine. Compared with the existing HPLC-FD or immunoassay methods, the proposed strategy presents the most appropriate balance between accuracy and facility, resulting in a considerable improvement of real-time quality control, and thereby, preventing chronic poisoning caused by OTA contained red wine.