In order to evaluate the functional characteristics of Tibetan kefir, the conditions for the fermentation of skim milk with Tibetan kefir were preliminarily optimized by one-factor-at-a-time method based on titratable acidity and sensory evaluation, and the antioxidant capacity and cholesterol-lowering ability of the fermented milk were studied in vitro. It was found that the optimal fermentation conditions were as follows: inoculum size 6%, fermentation time 7.5 h, and temperature 37 ℃. The fermented milk produced under the optimized conditions had a medium acidity, delicate taste, uniform texture and unique flavor, and its lactic acid bacterial population was 1.48 × 108 CFU/mL. The hardness of the fermented milk was 178.43 g, higher than that of traditional fermented milk, the adhesion was -103.52 g·s and the non-fat solid content, protein content and titratable acidity all conformed to the national standard for fermented milk. The quality of the fermented milk was good overall. Compared with traditional fermented milk, its antioxidant activity was not particularly prominent, but the cholesterol-lowering ability was particularly considerable, with removal rate of as high as 37.98% (fermented skim milk) and 40.68% (fermented whole milk).