Currently research of lactic acid bacteria focus primarily on the functional probiotics, which are major beneficial biota in the gastrointestinal tract, have been industrial manufactured. Probiotics confer health benefits on the host need adequate amounts. However, the absence of data makes it difficult to ensure the maintenance biological activities and population of probiotic. In this research, a fractional factorial design and steepest ascent experiment were used to analyze the influence of lyoprotectant as carbohydrates, prebiotics and amino acids on the survival of the probiotic Lactobacillus rhamnosus. The results indicated a maximum survival rate and population of viable bacteria of L. rhamnosus to be 55.84 % and 1.60 ×10¹¹ CFU/g after freeze-dried by using a combination of 10 g/100mL Sucrose, 2.5 g/100mL Isomaltooligosaccharide, 12 g/100mL Hydroxyproline. To a large extent, the survival and viability were dependent on the cryoprotectant used and make probiotics more attractive from a practical application in industrial viewpoint.