Abstract: To evaluate the effects of different starter cultures and different fermentation substrates on the quality of fermented milk, the samples were prepared with Bifidobacterium animalis subsp. lactis (Ba) in co-cultures with traditional yogurt starter cultures (SL), and the acidification profile, viable bacteria count, water-holding capacity, proteolytic activity, rheological property, physical stability, microstructure, volatiles and sensory characteristic of fermented cow milk, fermented yak milk and fermented milk mixtures of cow to yak milk ratios of 1:1 were investigated. The results showed that for the same fermentation substrates, Ba-SL significantly improved the acidification property and viable bacteria count of fermented milk (P<0.05), and increased the proteolytic activity of fermented cow milk, yak milk and mixed milk at the first day by 0.026, 0.025 and 0.016 compared with SL alone, respectively. Furthermore, Ba-SL improved the gel network structure, volatiles, and sensory quality of fermented milk. However, the co-cultures of Ba-SL had no significant effect on the physical stability of fermented milk (P>0.05). Ba-SL significantly enhanced the water-holding capacity of fermented cow milk and fermented mixed milk (P<0.05), and there was a higher water-holding capacity in fermented mixed milk in comparison to fermented cow milk and fermented yak milk during the whole refrigeration period, with the largest values of water-holding capacity being 45.68% on the 7 d. SL starter in conjunction with Ba increased the viscoelasticity of fermented yak milk, but decreased the viscoelasticity of fermented cow milk and fermented mixed milk compared to the single-culture of SL. For the same starter cultures, the acidification property, viable bacteria count, physical stability, and sensory score of fermented cow milk were significantly higher than those of the other groups (P<0.05), and the viscoelasticity of fermented yak milk was the highest among the fermented milk. The water-holding capacity of fermented mixed milk was significantly higher than those of the other groups throughout the refrigeration period (P<0.05), and the gel network structure and key volatiles of fermented mixed milk was the densest and more diverse among the three fermented milk, while the proteolytic activity of fermented mixed milk was between fermented cow milk and fermented yak milk. This study suggested that Ba in co-culture with SL could improve the quality of fermented milk, and both of cow milk and mixtures of cow and yak milk were more suitable to prepare fermented milk compared to yak milk.