To establish a simple，sensitive，accurate and rapid detection method for bifidobacteria. Molecular beacon probe and primers were designed and sythesized according to the conserved gene of Bifidobacterium in GenBank，and then reaction parameters of real-time PCR were optimized. For Real-Time PCR and molecular beacon detection for bifidobacteria, the intra-assay and inter-assay coefficient of variation were both less than 5%, indicating good reproducibility of this method, and no non-specific amplification was observed either. Compared with conventional PCR, this method is more sensitive and faster, and the detection limit was 5.7fg/PCR for bifidobacteria DNA. A linear standard curve was obtained between 2×10⁸CFU/mL and 2×10⁴CFU/mL (R>0.97). The method of Real-time PCR and molecular beacon detection for bifodobacteria has many advantages, such as being sensitive, specific, simple and fast, and this method can be used in situ detection of bifidobacteria quantitatively.