In this study patulin was determined by HPLC on a reversed phase C18 column with UV detection at 276 nm. Patulin was extracted with ethyl acetate and then was cleaned up by extraction with sodium carbonate solution. The mobile phase and the column used in the HPLC determination were selected to separate patulin from the interfering substances in apple juice, especially 5-hydroxymethylfurfural (HMF). The linear regression coefficient of the standard curve (y = 327x 292) for patulin at concentrations ranging from 15 to 200 μg/L was 0.9998 (N = 6). The results were corrected for the recovery percentage. The method is rapid and convenient for the determination of patulin in apple juice.