Aflatoxin B₁ was known to have the strongest poisonous character among Aflatown compounds. To search the production of Aflatoxin B₁ according 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 B₁ shows that the highest production appeared at 25 ℃ and its relative humidity 70%. The result that was quantitatively analyzed by using HPLC was 23.35 ppm, and by using fluorescence detector was 23.50 ppm. Production of Aflatoxin B₁ shows that the lowest production appeared at 20 ℃ and its relative humidity 50%. The result that was Quantitatively analyzed by using HPLC was 6.26 ppm, and by using fluorescence detector was 6.33 ppm. The structure of Aflatoxin B₁ was identify with authentic sample by using instrumental analysis methods of MS, ¹H-NMR, FT-IR, respectively.