[Background] Lactobacillus sp. is an important function microorganism in Chinese liquor fermentation. However, there is no quantitative method to uncover the distribution of Lactobacillus sp. among different production locations. [Objective] This study aimed to build a quantitative method to uncover the distribution Lactobacillus sp. among different Chinese liquor production locations. [Methods] Specific primers were designed based on 16S rRNA gene sequence, and the specificity was verified by PCR. The qPCR reaction procedure was further optimized to increase amplification efficiency and reveal the distribution of Lactobacillus sp. [Results] a pair of specific primers were designed and validated, its amplification product size was 445 bp. By optimizing annealing and extension conditions, a three-step qPCR quantitative method was developed with independent denaturation, annealing and extension process. The amplification linearity of this method was strong (R2>0.99). The sensitivity was high, the quantitative limit was 17.9 copies/μL. The repeatability was well, the coefficient of variation of Ct value was less than 1%. Following and detecting the representative fermentation system from 10 producing locations in China, Lactobacillus sp. presented in 8 producing areas. In the different making process samples from the same region, Lactobacillus sp. was detected but its content was significantly different. The content of Lactobacillus sp. from sesame aroma fermentation system in Weifang, Shandong province was the highest (7.27±0.04 lgcopies/g). The growth curve of Lactobacillus sp. in this system was divided into two stages: the growth stage (0 to 15 d) and the stable stage (15 to 45 d). [Conclusion] The established novel qPCR method can accurately quantify and identify Lactobacillus sp. Through follow-up detection, it was found that Lactobacillus sp. was widespread in Chinese liquor fermentation system, the distribution of Lactobacillus sp. was determined by the origin, the content of Lactobacillus sp. was affected by the making process. During fermentation process, the content of Lactobacillus sp. has an obvious dynamic characteristic. This study will improve the development of function study about Lactobacillus sp.