Foxtail millet is a kind of functional food rich in folic acid, and it is popular in the northern China. However, there are significant differences in folic acid contents in crop grains, such as the determination method and content analysis of folate in Foxtail millet, which make it difficult to identify the quality of millet and select the germplasm with high folate content. In this study, ‘triple-enzymes method’ was used to isolate folic acid from foxtail millet grains and construct a large-scale detection method by HPLC for folic acid content. Meantime, the folic acid contents were assessed among 45 accessions of foxtail millet in 2016 and 2017. The results showed that: based on HPLC, a folic acid content determination method for foxtail millet was successfully established. Folic acid standard curve showed the linear correlation between folic acid standard and peak area value, the correlation coefficient was 0.99. The relative standard deviation (RSD) was used to evaluate the precision, stability and repeatability in the detection process of folic acid content, and they were 0.81%, 1.19% and 1.65%, respectively. The range of grain folic acid content of 45 millet varieties were 0.26 ~ 2.56 μg/g in 2016 and 0.53 ~ 2.86 μg/g in 2017, with average values of 1.19 and 1.84 μg/g, standard deviation of 0.47 and 0.51 μg/g, and variation coefficient of 39.5% and 27.7%, respectively. The gaussian distribution had a little skewness. Finally, 9 high folic acid (folate content≥1.91 μg/g) and 3 low folic acid (folate content≤1.15 μg/g) accessions were screened from 45 millet accessions, respectively. This study not only provided the standard determination and evaluation system of folate in foxtail millet, and also laied a foundation for the innovation and utilization of high folate germplasm of foxtail millet.