Abstract: Pichia angusta can use ¹³C methanol as a sole source of carbon and synthesize ¹³C trehalose through fermentation. Some conditions on trehalose fermentation and extract were explored through uniform design. The fermentation conditions as follows: thalli was incubate for 20 h at 37 ℃, and then elevated temperature to 44 ℃, continued to incubate for 1 h. The extract conditions was as follows: the concentration of ethanol was 50%, the temperature of extract was 42 ℃, the time of extract was 110 min, the ratio of material and liquid was 1∶30. The product abundance and purity were more than 98% which was obtained using 99% abundance ¹³C methanol. Almost no dilution of the label substance occurred.