The invention discloses a preparation method of Gracilaria agar oligosaccharide promoting growth of Lactobacillus bulgaricus. The preparation method includes following steps: step 1, removing impurities of Gracilaria: cleaning, removing impurities of Gracilaria, drying, and placing Gracilaria in water bath at 80 DEG C for heating for 2 h; step 2, sterilizing at high temperature: sterilizing at 121 DEG C for 20 min; step 3, squeezing glue: using filter cloth to wrap Gracilaria, squeezing, collecting a polysaccharide solution, cooling, drying, smashing into granules, and bagging the granules for standby use; step 4, performing enzymatic treatment: dissolving cooled coarse polysaccharide in water to prepare a polysaccharide solution of 5g / L in concentration, adding agarase for treatment, performing enzymolysis for 20-1280 min to prepare an oligosaccharide solution, wherein enzyme adding quantity is 15% and enzyme activity is 300 U / mL; step 5, drying: freeze-drying the oligosaccharide solution or vacuum-drying the same to form solid powder to obtain the agar oligosaccharide. The preparation method can be used for Lactobacillus fermentation enterprises and dairy product production enterprises to produce high-activity Lactobacillus products and high-storability dairy products.