Aflatoxin B₁was known to have the strongest poisonous character among Aflatoxn compounds. To search the production of Aflatoxin B₁according to the change of temperature and relative humidity, millet incubated with Aspergillus flavus KCCM 35078 was incubated at 20℃, 25℃, 30℃, 25℃ and at relative humidity 50%, 60%, 70% and for incubation time 72, 96, 120, 144, 168, 192 hours. Production of Aflatoxin B₁shows that the highest production appeared at 25℃ and its relative humidity 70%. The result that was quantitatively analyzed by using HPLC was 23.35ppm, and by using fluorescence detector was 23.50ppm Production of Aflatoxin B₁shows that the lowed production appeared at 20℃ and its relative humidity 50%. The result that was Quantitatively analyzed by using HPLC was 6,26ppm, and by using fluorescence detector was 6.33ppm The structure of Aflatoxin B₁was identify with authentic sample by using instrumental analysis methods of MS, H-NMR, FT-IR, respectively.