In this study, a combination of an improved mucin enriched medium with real-time polymerase chain reaction (real-time PCR) was used to test 48 samples of maternal and infant feces for Akkermansia muciniphila (Akk). Under optimized conditions, 24 Akk strains were isolated from eight positive samples. All these strains were confirmed as Akk by 16S rRNA gene sequencing and PCR with Akk-specific primers. Repetitive extragenic palindrome-polymerase chain reaction (rep-PCR) fingerprinting classified the 24 strains into four genotypic groups. Subsequently, these strains were tested in vitro for simulated gastrointestinal fluid tolerance, hydrophobicity, antibiotic susceptibility, and glycan utilization capacity. The results showed that strains HN18D-1, HN18D-3, and WW48D1-13 had the highest tolerance to simulated gastric and intestinal fluids. All Akk strains were resistant to vancomycin, clindamycin, kanamycin and erythromycin. Xylooligosaccharides and soybean oligosaccharides had prebiotic effects on the Akk strains. Collectively, Akk isolates HN18D-1, HN18D-3 and WW48D1-13 can be used as potential probiotic candidates for subsequent in-depth studies.