[Background] The probiotic functions of Bifidobacterium are widely recognized, and a large body of studies paid attention to the biodiversity of Bifidobacterium in the intestine. However, Bifidobacterium is a low abundance genus in the intestine, and it is difficult to study the bifidobacterial diversity by extant technologies in depth. [Objective] To screen a pair of specific primers for the analysis of diversity of Bifidobacterium with low abundance in fecal samples. [Methods] Initially, to obtain Bifidobacterium-specific primers with amplicons of over 800 bp, the primers were recombined and optimized according to the relative positions of the extant bifidobacterial primers and their matching rates with the Bifidobacterium 16S rRNA gene sequence. Then, the rest primers were screened by PCR amplification and agarose gel electrophoresis, and their specificity was verified. Finally, taking the universal bacterial primer (27f/1492r) as control, the DNA amplicons of bacterial microbiota in three fecal samples amplified by selected primers were sequenced by SMRT (Single-molecule real-time) sequencing technology, and different sequencing primers were compared and analyzed at the species level. [Results] Two pairs of primers, Bif164-f/Pbi R2 and Pbi F1/Pbi R2, were selected as the optimal Bifidobacterium primers theoretically with amplicon greater than 800 bp from 9 pairs of Bifidobacterium-specific primers collected from literature. The PCR amplification and agarose gel electrophoresis showed that the amplified bands of Bif164-f/Pbi R2 were bright and no tail. In addition, sequencing and analysis of DNA amplicons from bacterial microbiota in three fecal samples with primers 27f/1492r and Bif164-f/Pbi R2 using SMRT sequencing platform. The analysis of 27f/1492r amplicons showed that three samples contained 1, 3 and 4 bifidobacterial species respectively, and the average relative content of Bifidobacterium was 0.34%. The analysis of Bif164-f/Pbi R2 amplicons showed that three samples contained 2, 6 and 8 bifidobacterial species respectively, and the average relative content of Bifidobacterium was 98.72%. The above results indicate that Bif164-f/Pbi R2 can specifically detect bifidobacteria with low abundance in feces at species level, which realize the diversity analysis of Bifidobacterium in samples. [Conclusion] In summary, a pair of Bifidobacterium-specific primers Bif164-f/Pbi R2 were screened in the experiment, which can be used to analyze the diversity of low-abundance Bifidobacterium in fecal samples at species level. It was also confirmed that the combination of theory and experiment is feasible to perform primers screening.