Abstract: In order to establish a rapid lipid determination method in Yarrowia lipolytica, the determination conditions were studied with the Nile Red fluorescence spectrometry. The optimal determination conditions were confirmed by researching the optimal emission wavelength in Yarrowia lipolytica, optimal volume fraction, dosage of Nile Red dye solution, dyeing time and efficiency of different penetrating agents, the linearity between lipid content in cell and fluorescence intensity were affirmed.Yarrowia lipolytica had maximum fluorescence value in excitation wavelength 560 nm, emission wavelength 650 nm, 20 μL0.1 mg/m L mass fraction of Nile Red dye solution and 15% volume fraction of isopropanol were added in per milliliter of Yarrowia lipolytica suspension respectively, dyeing time 5 min in darkness, in the range of cell with OD600 of 0¹.3, there was a good linear relationship between lipid content ( X) of solution and fluorescence intensity ( Y) , and the linear formula was Y =6.3651 X + 10.097 ( R2= 0.9902) , the sensitivity of this method was 0.0001 g. The Nile Red fluorescence spectrometry could reflect the lipid content accurately in Yarrowia lipolytica and be used as a new method for rapid lipid determination in Yarrowia lipolytica.