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. Peppers 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 18.61ppm, and by using fluoroscence detector was 19.39ppm. Production of Aflatoxin B1 shows that the lowest production appeared at 20℃, its relative humidity 65% and 4days. The result that was quantitatively analyzed by using HPLC and fluoroscence detector was 0.19ppm same value. 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.