An extraction method based on molecularly imprinted polymer prepared through a mimic template approach was used for the determination of ochratoxin A in 17 red wines from different geographical regions of Italy. Sample loading (wine sample diluted 1:1 with 1% v/v aqueous solution of PEG 8000), washing (2 mL water/acetonitrile 4:1 v/v), and elution (2 mL of acetonitrile/acetic acid 98:2 v/v) conditions allowed the optimization of the extraction method, capable of preconcentrating ochratoxin A below the maximum permitted level of 2 ng/mL. Under optimized conditions, recoveries of ochratoxin A from spiked samples ranged from 88 to 102% with sample volumes up to 20 mL. The HPLC determination by fluorescence detection allowed limits of detection and quantification, respectively, of 0.075 and 0.225 ng/mL. Sample extractions by an immunoaffinity protocol showed the method to be comparable, demonstrating the potential of the imprinting approach to substitute for the current immunoaffinity method.