Air susu ibu (ASI) mengandung immunoglobulin, sitokin, dan berbagai enzim pencernaan serta mengandung prebiotik yang disebut Human Milk Oligosaccharides (HMO) yang merupakan nutrisi bagi mikrobiota saluran pencernaan bayi seperti Bifidobacterium spp. Penelitian ini bertujuan untuk mengidentifikasi dan mengkarakterisasi isolat Bifidobacterium sp. BS2-PS1 dari ASI sebagai kandidat probiotik. Metode penelitian ini mencakup purifikasi dan pemeliharaan isolat BS2-PS1; uji identifikasi morfologi dilakukan dengan pewarnaan Gram dan endospora; karakterisasi biokimia dengan uji katalase; identifikasi molekuler dengan 16S rRNA sequencing; dan karakterisasi sifat probiotik dengan uji ketahanan terhadap phenol (0,2% dan 0,5%; 24 jam), NaCl (1,5%, 2,5%, 3,5%; 24 jam), simulated gastric juice (pH 2; 0, 30, 60, dan 120 menit), dan garam empedu (0,3% dan 0,5%; 1, 2, 3 jam). Hasil dari penelitian ini isolat BS2-PS1 memiliki karakteristik koloni berwarna putih, cenderung membentuk koloni bulat kecil, margin entire, konveks, terlihat smooth dan berkilau serta memiliki bentuk sel bifid dengan penebalan pada ujung sel; pewarnaan Gram menunjukkan BS2-PS1 adalah Gram positif dan endospora negatif; karakterisasi biokimia menunjukkan katalase negatif; identifikasi 16S rRNA menunjukkan isolat BS2-PS1 memiliki 99.48% kesamaan dengan spesies Bifidobacterium breve; analisis sifat probiotik menunjukkan bahwa uji terhadap phenol konsentrasi 0,2% dan 0,5% didapat survival rate masing-masing 6,53% dan 6,36%, uji terhadap NaCl konsentrasi 1,5%, 2,5%, dan 3,5% diperoleh survival rate masing-masing 91%, 74%, dan 67%, uji terhadap simulated gastric juice (pH 2; 30, 60, dan 120 menit) diperoleh survival rate masing-masing 92,1%, 84,3%, dan 78,1%, uji terhadap media garam empedu (0,3% dan 0,5%, pada 0, 1, 2, 3 jam), didapat nilai viabilitas CFU/mL (106) masing-masing pada 10,6; 11,8; 15,8; 27,4 (0,3%) dan 11,3; 14,4; 11,4; 13,4 (0,5%). Penelitian ini dapat disimpulkan dari beberapa analisis dasar probiotik bahwa B. breve BS2-PS1 memiliki potensi untuk dikembangkan sebagai probiotik. / Breast milk (ASI) contains immunoglobulins, cytokines, and various digestive enzymes and contains prebiotics called Human Milk Oligosaccharides (HMO) which are nutrients for the microbiota of the baby's digestive tract such as Bifidobacterium spp. This study aims to identify and characterize Bifidobacterium sp. isolates. BS2-PS1 from ASI as a probiotic candidate. This research method includes purification and maintenance of BS2-PS1 isolates; morphological identification test was carried out by Gram staining and endospores; biochemical characterization with catalase test; molecular identification by 16S rRNA sequencing; and characterization of probiotic properties by testing resistance to phenol (0.2% and 0.5%; 24 hours), NaCl (1.5%, 2.5%, 3.5%; 24 hours), simulated gastric juice (pH 2; 0, 30, 60, and 120 minutes), and bile salts (0.3% and 0.5%; 1, 2, 3 hours). The results of this study isolate BS2-PS1 has the characteristics of white colonies, tends to form small round colonies, entire margins, convex, looks smooth and shiny and has a bifid cell shape like the letter "Y" with thickening at the ends of the cells; Gram staining showed BS2-PS1 was Gram positive and endospores negative; biochemical characterization showed negative catalase; identification of 16S rRNA showed isolate BS2-PS1 had 99.48% similarity with Bifidobacterium breve species; Analysis of probiotic properties showed that tests on phenol concentrations of 0.2% and 0.5% for 24 hours obtained survival rates of 6.53% and 6.36% respectively, tests on NaCl concentrations of 1.5%, 2.5% and 3.5% for 24 hours obtained survival rates of 91%, 74% and 67% respectively, tests on simulated gastric juice (pH 2, at 30, 60 and 12 0 minutes) obtained a survival rate of 92.1%, 84.3% and 78.1% respectively, tests on bile salt media (0.3% and 0.5%, at 0, 1, 2, 3 hours), obtained CFU/mL viability values (10-6) respectively at 10.6; 11.8; 15.8; 27.4 (0.3%) and 11.3; 14.4; 11.4; 13.4 (0.5%). From this study it can be concluded from several basic analyzes of probiotics that B. breve BS2-PS1 has the potential to be developed as a probiotic.