In this study, aflatoxin B1 (AFB1) aptamers were covalently coupled on the surface of magnetic beads to prepare aptamer affinity magnetic beads (AAMBs). Furthermore, the AAMBs, incubation solution, washing solution and eluate were pre-encapsulated in reagent strips to develop a single sample pretreatment kit of AFB1. The automatic pretreatment of AFB1 was realized through the automatic mycotoxin purifier. The content of AFB1 in the eluate was determined by ultra high performance liquid chromatography (UPLC). The preparation conditions of AAMB and the use conditions of AFB1 single sample pretreatment kit were determined by optimizing the particle size of magnetic beads, the indirect arm length of aptamer, the amount of AAMB, and the volume of incubation solution. Recoveries was compared by manual and automatic pretreatment methods. Under the optimized conditions, the limit of detection (LOD) was 25 pg/mL, and the spiked recoveries at low, medium and high levels in corn and wheat ranged from 92.8% to 110.1%, with the relative standard deviation (RSD) of intra-day precision less than 5.1% and the RSD of 6.7% in term of inter-day precision, which showed good sensitivity, accuracy and repeatability. The results of AFB1 in the national certified reference materials of grain and oil substrates (corn, brown rice, peanut oil and peanut butter) were within their extended uncertainty range. The single sample pretreatment kit developed could automatically complete the pretreatment of 10 samples within 20 min with the aid of mycotoxin automated purification instrument. This method has the advantages of short time, high throughput, small batch-to-batch variation and no need of cold chain storage and transportation. It has a good application prospect in the quantitative detection of mycotoxins, and could lay a foundation for the development of automatic pretreatment methods for other targets.