With the rapidly development of fermented milk product, it is significant for enhancing the performance of starter culture. This paper not only investigated the influence of anti-freeze factors and freeze-drying protective agents on viable count, freeze-drying survival rate and yield of Lactobacillus bulgaricus (LB) and Streptococcus thermophilus (ST), but also optimized the bacteria proportion of freeze-dried starter culture for yogurt by full factorial experimental design. The results showed as following: the freeze-drying protective agents or anti-freeze factors could enhanced survival rate of LB and ST; the freeze-dried LB and ST powders containing both of anti-freeze factors and freeze-drying protective agents had higher viable count and freeze-drying survival rate that were 84.7% and 79.7% respectively; In terms of fermentation performance, the best group of freeze-dried starter for yogurt was the compound of LB3 and ST2.