Abstract: The effects of high density fermentation Kluyveromyces marxianus on nutritional component and cultivation condition of culture medium was studied. Single factor experiment revealed that YPD as the basic medium for the proliferation of Kluyveromyces marxianus. When the medium was 67.37 g/L sucrose, yeast extract 29.7 g/L, corn steep 15.61 g/L, KH₂PO₄ 4.13 g/L, MgSO₄0.3 g/L, initial pH6, fermentation temperature was 30 ℃, stirring speed was 160 r/min, the fermentation biomass of 18 h Kluyveromyces marxianus was ( 9.34 ± 0.12) g/L by response surface analysis and culture condition orthogonal test.Fermented dairy experiments showed that there was no obvious difference in bacterial biomass, taste and flavor of fermented dairy produced by high density and skimmed milk medium culture Kluyveromyces marxianus.Therefore, the optimized high density fermentation medium formula and process conditions were suitable for the cultivation of the yeast Kluyveromyces marxianus.