The objective of present research was to study the effects of fat addition level and different ratios of fat sources on nutrient apparent digestibility and serum biochemical indices of Holstein steers. Four (2. 50 ± 0.17)⁃year⁃old Holstein steers [average body weight of (580±50) kg] with permanent rumen fistula were fed a basal diet consisting of corn silage and concentrate supplement, which was added with 2 sources of fat (calci⁃ um salts of flaxseed fatty acids vs. whole flaxseed) at 3 addition levels (4%, 5% and 6%) and with 4 differ⁃ ent ratios (100 / 0, 85 / 15, 70 / 30 and 5 / 45) in a 3 × 4 two⁃factorial design. Fat addition level / ratio in the twelve groups were 4 / 100, 4 / 85, 4 / 70, 4 / 55, 5 / 100, 5 / 85, 5 / 70, 5 / 55, 6 / 100, 6 / 85, 6 / 70 and 6 / 55, respectively. The experiment was divided into 12 stages, and the first 14 d of each stage was an adjustment pe⁃ riod and the following 5 d was for sample collection. The results showed as follows: 1) the apparent digestibili⁃ ties of crude protein and acid detergent fiber in 4% and 5% fat group were very significantly higher than those in the 6% fat group (P<0.01). 2) The activities of serum fatty acid triglyceride lipase, carnitine palmitoyl transferase⁃Ⅰ and 3⁃hydroxy⁃3⁃methyl⁃glutaryl coenzyme A reductase in 5% and 6% fat groups were very sig⁃ nificantly higher than those in the 4% fat group (P<0.01). The contents of serum total protein and very low density lipoprotein cholesterol in 6% fat group were significantly higher than those in 4% fat group (P<0.01 or P<0.05). The content of serum total protein in 70 / 30 ratio group was significantly higher than that in other ra⁃ tio groups (P<0.05). The content of serum high density lipoprotein cholesterol (HDL⁃C) in the 5% and 6% fat groups was significantly higher than that in the 4% fat group (P<0.01), the content of serum low density lipoprotein cholesterol (LDL⁃C) in the 4% fat group was significantly higher than that in the 6% fat group (P<0.05). In conclusion, based on data of nutrient apparent digestibility and serum biochemical indices, the optimum fat addition level is 4% to 5%, and the optimum of different fat source ratios (rumen undegradable unsaturated fat / flaxseed) is 70 / 30.