Patulin contamination in fruits, vegetables, and their products is considered a serious health risk factor for food safety and human health. Thus, a rapid, simple detection method for patulin is becoming important, which could provide a tool for routine screening and food surveys. The objective of this study was to develop a sensitive aptamer-based lateral flow assay (FLA) using Streptavidin functionalized gold nanoparticles for sensitive patulin detection. An excellent dynamic range for patulin detection was obtained (2.7~139.8 ng/mL in the buffer and 7.07~359.5 ng/mL in the sample) with no affinity for other mycotoxins such as zearalenone (ZEN), ochratoxin A (OTA), aflatoxin B1 (AFB1), citrinin or tenuazonic acid (TEA). The limit of detection was 0.19 ng/mL in the buffer and 0.36 ng/mL in the real sample. The recoveries were 83.3% to 107.1%, with a satisfactory RSD value from 6.5% to 7.5%. Hence the established LFA could be used as a rapid, simple, on-site screening tool for PAT determination in apple juice.