It was accurately analyzed to the amount of production according to the change of temperature, relative humidity and time of Aflatoxin B1 on the strongest poisonous character among Aflatoxin compounds. Licorice inoculated with Aspergillus parasiticus KCCM 35078 was incubated at 20℃, 25℃, 30℃, 35℃ and at relative humidity 65%, 70%, 75% and for incubation time 96, 120, 144, 168, 192 hours. Production of Aflatoxin B1 shows that the highest production appeared at 30℃, its relative humidity 70% and 8days. The result that was quantitatively analyzed by using HPLC was 24.29ppm, and by using fluoroscence detector was 25.05ppm. Production of Aflatoxin B1 shows that the lowest production appeared at 35℃, its relative humidity 75% and 4days. The result that was quantitatively analyzed by using HPLC was 0.54ppm and by using fluoroscence detector was 0.57ppm. The structure of Aflatoxin B1 was identify to agreement with authentic sample by using instrumental analysis methods of FT-IR, 1H-NMR, GC/MS respectively.