Abstract: Bifidobacterium is a typical intestinal probiotics of strictly anaerobic, and studying its probiotic properties is very important for human health. Through 16S rRNA sequencing and phylogenetic tree construction, a strain of Bifidobacterium longum with certain oxygen tolerance was isolated and identified (named Bifidobacterium longus F16). In addition, through morphological identification and physiological biochemical identification, this strain has the following characteristics:neat colony edge, convex, smooth and opaque, milky or gray surface, catalase is negative, fructose-6-phosphate phosphatase is positive, the strain can decompose lactose, fructose, maltose, and produce acid. By means of HPLC, colorimetric method, plate counting method, and simulated gastrointestinal environment, the content of lactic acid, the ability to degrade cholesterol (1% and 5% inoculum), the acid resistance, bile salt resistance and the tolerance to artificial gastrointestinal fluid of this strain were studied. The concentration of lactic acid produced by the strain was 10.63 mg/mL. The degradation rate of cholesterol was between 46-75%, suggesting that a significant cholesterol degradation effect, and this degradation was in a time-dependent manner. The tolerance curve to acidity was more gentle, the acid resistance was better, and the survival rate was also high (85.20%) in simulated gastrointestinal simulation environment. At 0-3% bile salt concentration, the tolerance was stronger than that of strictly anaerobic Bifidobacterium. As a result, F16 has excellent probiotic properties and has important application value for the development of a new generation of Bifidobacterium products.