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 and relative humidity, apple kinds incubated with Aspergillus parasiticus KCCM 35078 sas incubated at 20°C, 25°C, 30°C, 35°C and at relative humidity 65%, 70%, 75%, 80% and for incubation period 4, 5, 6, 7, 8 days. As a result of having confirmed the production of Aflatoxin B1 generated in this study, apple kinds did not have a lot of the production of Aflatoxin B1. Production of Aflatoxin B1 shows that the highest production appeared at 20°C, its relative humidity 70% and for incubation period 8days. The result that was quantitatively analyzed by using HPLC was 20.62ppm, and by using Fluoroscence detector was 25.75ppm. Production of Aflatoxin B1 shows that the lowest production appeared at 35°C, its relative humidity 70% and for incubation period 4days. The result that was quantitatively analyzed by using HPLC was 0.23ppm, and by using Fluoroscence detector was 0.25ppm. The structure of Aflatoxin B1 was confirmed to identify with authentic sample by using instrumental analysis methods of GC/MS, 1H-NMR, FT-IR respectively.