Abstract: To explore the optimum conditions for the preparation of Ganoderma lucidum oligosaccharides by enzymatic hydrolysis and ultrasonic technology and the proliferation of oligosaccharides on Lactobacillus were studied. Conditions of enzymatic hydrolysis and ultrasonic treatment of Ganoderma lucidum aqueous extracts were optimized. Yields and structures of these oligosaccharides produced by 3000 Da ultrafiltration membrane were compared and analyzed, and the proliferation effect on Lactobacillus in vitro was investigated. Results showed that under the enzyme hydrolysis conditions of 9 h, temperature 50 ℃, pH5, enzyme dosage 1000 U/g substrate, ultrasonic time 60 min and ultrasonic power 480 W, the yield of Ganoderma lucidum oligosaccharides was the highest, about 1.76%, which was 61.46% higher than that in the raw materials. It demonstrated that Ganoderma lucidum oligosaccharides had typical characteristics of β-glycoside bond and pyran ring compounds by FTIR. Ganoderma lucidum oligosaccharides had a good proliferation effect on Lactobacillus, and better than that of Ganoderma lucidum polysaccharides. This study would provide a basic technology for preparation of Ganoderma lucidum oligosaccharides and expand its application in the development of functional foods.