To evaluate the influence of grinded particle size on the determination of aflatoxin B1(AFB1), improve the sample preparation method and the repeatability of the current national standard detection method for the determination of AFB1 of corn raw grain samples, this study uses natural AFB1-positive corn as experimental material, and four grades of samples with different particle size ranges were obtained by means of graded preparation, so as to maximize the differences of particle size distribution of samples at all levels. The influence of sample preparation on AFB1 determination values was evaluated by AFB1 determination values and coefficient of variation in samples of all levels, and the optimal sample preparation requirements and sample size were obtained and verified. The AFB1 content of different particle size samples was inversely proportional to that of the sample after grinding. When the sample weight is 5 g, it should be crushed until all through the 0.6 mm sieve. When the sample weight is 10 g, it should be crushed until all through the 0.9 mm sieve, so that the test results meet the requirements of GB/T 32465—2015《Requirements for verification & validation of detection methods and internal quality control on chemical analysis》. The results give consideration to the determination accuracy and operation efficiency of AFB1, which can be applied to the detection of other mycotoxins and has important significance for improving the detection methods of mycotoxins in foodstuff.