Aflatoxin B₁ was known to have the strongest poisonous character among Aflatoxin compounds. To search the production of Aflatoxin B₁ according to the change of temperature, relative humidity and incubation days, orange incubated with Aspergillus parasiticus KCCM 35078 was incubated at 20℃, 25℃, 30℃ at relative humidity 60%, 70%, 80% and for incubation time 3, 4, 5, 6, 7 days.. 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 77.41ppm, and by using Fluorosccence detector was 87.87ppm. Production of Aflatoxin B₁ shows that the lowest production appreared at 20℃ and its relative humidity 80℃(HPLC), 60%(Fluoroscence detector). The result that was quantitatively analyzed by using HPLC was 4.47ppm, and by using Fluoroscence detector was 4.17ppm. The structure of Aflatoxin B₁ was identify with authentic sample by using instrumental analysis methods of GC/MS, ¹H-NMR, FT-IR respectively.