The carbohydrate, prebiotics/alcohols as well as protein/amino acid have shown important influence on the cryoprotectants for Streptococcus thermophiles, this study aimed to screen the main effective factors forcryoprotectants of Streptococcus thermophiles using Plackett-Burman design. Viable counts with cryoprotectants were studied containing eight carbohydrate (glucose, fructose, sucrose, lactose, galactose maltose, trehalose, soluble starch), eleven prebiotics/alcohols (xylooligosaccharide, fructo-oligosaccharide, galactooligosaccharide, isomaltooligosaccharide, stachyose, synanthrin, dextran sulfate, glycerin, sorbitol, mannitol, inositol) and eight protein/amino acid (peptone, yeast powder, casein hydrolysate, glutamic acid, methionine, cysteine, alanine, Vc). The results indicated that sucrose and soluble starch, dextran sulfate, casein hydrolysate among the carbohydrate, prebiotics/alcohols and protein/amino acid affected the cryoprotectants for Streptococcus thermophiles markly, which should be appropriately increased in the subsequent experiments.