Given severe health-hazardous effects of aflatoxin B1 (AFB1) widely occurring in cereal grains and animal feeds, it is highly urgent to develop analytical methods for its rapid screening. In this work, we proposed a simple and high-throughput method for the determination of AFB1 in millet and buckwheat samples using high performance thin layer chromatography (HPTLC) linked to fluorescence densitometry. The first step was to optimize the solid-liquid extraction for the crude clean-up of the samples. The QuEChERS (Quick, Easy, Cheap, Effective, Robust and Safe) extraction strategy was used and different solvent systems for their extraction efficiency of AFB1 from the samples were evaluated. Then, trichloromethane/ethyl acetate (7/3, v/v) was used as the mobile phase to realize the separation of the targeted compound from background noises on silica gel plates. Quantification was readily performed with densitometry in fluorescence mode. In order to fix the optimal excitation wavelength, spectra scanning from 250-400 nm was carried out, revealing that 364 nm light gave the highest signal. With the optimized optical system, high sensitivity to AFB1 was achieved, with a limit of detection (LOD) at 3 μg/kg. Apart from that, good linearity (0.999) was obtained within the range of 1-80 ng/band of AFB1. To assess the analysis accuracy, two levels of AFB1 (50 and 100 μg/kg) were spiked into real grain samples. The obtained results showed that the recovery rates were within the range of (81.6–114.0)%. The proof-of-concept results of this work evidenced that HPTLC is a promising analytical tool for the screening of mycotoxin in difficult samples.