It is crucial to identify the distribution of lactic acid bacteria (LAB) in breast milk which is a source of live probiotics for the infant gut. Quantitative PCR (qPCR) is a powerful technique for selectively counting LAB at the species or strain level. However, the limit of quantification (LoQ) of qPCR is inadequate for human milk samples. To address this issue, nested primer pairs for and species were designed to enable 15 cycles of preamplification Subsequent qPCR assays using the preamplification products (with 10-fold dilution) as templates reduced the LoQ for enumeration plasmids to one-thousandth of the original concentration. Importantly, preamplification did not enhance the detection capability for low biomass samples because the efficiency of DNA extraction was too low. To mitigate the variation in bacteria concentration, inert bacteria were added to the samples. Incorporating at 10 CFU mL into the samples helped expand the linear range between the cycle threshold values and the concentration of target bacteria. By combining preamplification with inert , probiotic LAB cells could be detected at single-digit levels in breast milk, while also minimizing the requirements for primer quality and DNA extraction kits. This qPCR-based approach is a reliable tool for studying the potential distribution patterns of LAB in low biomass samples, and it enabled approximately a tenfold increase in the number of breast milk samples in which and species were detected, indicating the ubiquitous and in breast milk.