Abstract: This study established a two-stage fermentation process through the response surface optimization for the value-added utilization of the whey from the cheese production. In the study, a mathematical model was designed by use of Box-Behnken method, in which the alcohol content of the mash as the response value and the fermentation temperature, total inoculum, the fermentation time and initial pH as the dependent variable. Then the final product wine was evaluated and analyzed according to the various physical and chemical indicators such as amino acid and organic acid content. The result indicated that based on the two-stage fermentation process the lactose content in whey was 0.8% and the utilization rate of lactose reached 92.7% by inoculation of Kluyveromyces lactis first for 54 h, and then the full-bodied yak whey wine alcohol content reached 14.1% after inoculation Saccharomyces cerevisiae at pH5.5 and culture temperature 30 ℃ for 70 h with the total inoculation amount was 8% (Kluyveromyces lactis 4%). Finally, the full-bodied nutritious yak whey wine was produced by a two-staged brewing technology, which complied with national standards of the final essential amino acids, total amino acids and organic acids increased by 0.7 fold, 1.1 fold and 0.8 fold respectively, indacating that the fermented yak whey wine was a kind of milk wine with rich nutrition.