•SD-LLLME is applied to extract patulin for the first time.•ID-LC-MS method is developed for the analysis of patulin in apple juice.•Method is validated with fortified samples. Quick and simple analytical methodology has been developed for the measurement of patulin in apple juice by combining single-drop liquid-liquid-liquid microextraction with isotope dilution ultra-high performance liquid chromatography-mass spectrometry. After systematic parameters optimization, the whole sample pretreatment only consisted of single extraction that consumed 1.5 mL ethyl acetate. While, other methods usually needed complicated pretreatment, including extraction, purification, evaporation and redissolution. This sample pretreatment method could greatly lower the interferences from sugar-rich matrix, and the limit of detection was 0.5 μg/L and limit of quantification was 2 μg/L in apple juice. Moreover, linear range covered three orders of magnitude from 2 to 2000 μg/L. The proposed method is promising for patulin extraction and detection in apple juice, which will opens a new perspective in the enrichment of trace contaminations in high sugar complex matrix.