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Bifidobacterium longum is an important probiotic in the human and animal intestine, wherein both infant and long subspecies are edible strains with numerous uses in food and medicine. Here, we aimed to explore some significant phenotypes and potential genes involved in the safety and probiotic functions of Bifidobacterium longum subsp. infantis B2-01 isolated as well identified from the feces of 3-month-old infants in Guangzhou. The complete genome sequence showed that B. infantis B2-01 contained a 2,615,705 bp circular chromosome with a 59.22% G + C content, lacking plasmids. It featured 2149 coding genes in total. The detection of harmful metabolites, hemolysis test, antibiotic resistance test, and acute oral toxicity test in mice, combined with the analysis of genes involved in antibiotic resistance, virulence factors, and harmful metabolite production, we tentatively proved B. infantis B2-01 to be a safe strain. In vitro tolerance tests and genes involved in adaptive responses confirmed that B. infantis B2-01 has excellent gastrointestinal fluid tolerance, hydrophobicity capability, auto aggregation capability, adhesion capacity, antioxidant activity, and hypocholesterolemic activity, indicating that B. infantis B2-01 may be able to be a probiotic with high application value. • Complete genome of B. infantis B2-01 was firstly sequenced. • Systematic analysis of the safety and probiotics properties by genotype combined with corresponding phenotype. • Analyzed the oxygen stress genes in the genome. • A phylogenetic tree was constructed based on the core genome. [ABSTRACT FROM AUTHOR]