We established a rapid quantification method for detecting the absolute amount of total bacteria, Bifidobacterium, Enterococcus, and Enterobacteriaceae in gut microbiota. The recombinant plasmids for the target DNA fragments of each target bacterial taxa were constructed by molecular clone technique and used as a standard template for quantitative assays. The results showed the primers and probes were highly specific. The linearity of different bacterial standard curves had a high coefficient of determination (R²≥0.995). The linear range was 2.9×10³-2.9×10¹² copies/μL for all bacteria, 3.1×10²-3.1×10⁹ copies/μL for Bifidobacterium, 5.9×10²-5.9×10⁹ copies/μL for Enterococcus and 6.3×10²-6.3×10⁹ copies/μL for Enterobacteriaceae. The low threshold of copy number was 7.5×10²copies/μL for total bacteria, 46 copies/μL for Bifidobacterium, 37 copies/μL for Enterococcus, 51 copies/μL for Enterobacteriaceae, respectively. Moreover, the detection limit was 10 times higher than that of qPCR dye method. Besides, the coefficient of variation of the reproducibility experiment was below 1.32%，implying a well reproducibility. The method was used to detect clinical fecal samples of different ages, and the relationship between these bacteria account and ageing was analyzed, showing high consistent with those reported in other researches. In conclusion, a clinically applicable method for quantification of microbiota was established, which can detect 3 types of bacteria and total bacteria at the same time. And, it has some positive significance for the study of microbiome.