[Objective] To establish a simple method to screen oleaginous yeast and determine the intracellular lipid content. [Methods] The study is based on the theory that the combination of nile red and intracellular oil will emit fluorescence when induced by UV light and the fluoresence indensity is associated with the lipid content. We cultivate yeast in the culture medium added with nile red, and screen the oleaginous yeast strains from the 385 deep-sea yeasts by measns of obeserving the fluorescence of the yeast colony. We have identified the screened oleaginous yeast strains by the 26S rDNA D1/D2 series analysis method. Designating one of the oleaginous yeasts (2A00015) as the test strain, the lipid content rapid determination method by nile red dyeing was established. [Results] 22 oleaginous yeasts were obtained with the lipid content reaching up to 62.9%. Based on the molecular identification, it showed that the 22 yeasts are separately belong to Candida viswanathii, Candida parapailosis, Rhodotorla mucilaginosa, Debaryomyces hansenii, Pichia guilliermondii and Rhodosporidium paludi-genum. The optimum condition for lipid content determination by nile red dyeing is: bacterium suspension OD600 lower than 1.2, nile red concentration 0.5 mg/L, dyeing time 5 min, excit-ing wavelength 488 nm, emission wavelength 570 nm. The relative fluorescence intensity ob-tained by this method exhibits a positive association with the lipid content obtained by weigh-ing method, which can be explained as R2=0.9637.