To assess bacterial vaginosis (BV)-related primary molecular diagnostic markers of Lactobacillus crispatus, Gardnerella vaginalis, Fannyhessea vaginae, bacterial vaginosis-associated bacteria 2 (BVAB-2), Megasphaera-1 and Megasphaera-2 and to discover molecular diagnostic indicators of BV with the most economic value for the efficient diagnosis of BV.All vaginal secretion specimens, including 122 BV-positive cases and 130 BV-negative controls were collected. First, quantitative polymerase chain reaction (PCR) was used to determine the levels of above the six bacteria. Then, the detection rates, sensitivity, specificity, diagnostic threshold, and receiver operating characteristic (ROC) curve were compared. Megasphaera-1 and Megasphaera-2 were detected in the BV-positive group, with a low detection rate of 35.25% and 19.67% respectively. The sensitivity and specificity of the above four bacteria were 95.90%/72.31%, 82.79%/92.48%, 72.13%/95.38%, and 56.56%/94.62% respectively, using the cut-off value for the diagnosis of BV. When combinations of L. crispatus with G. vaginalis, F. vaginae, and BVAB-2 were performed respectively, their sensitivity and specificity were 99.29%/97.79%, 98.86%/98.72%, and 98.22%/98.51% in sequence.It is difficult to diagnose BV using only one species, however, combinations of L. crispatus with G. vaginalis or F. vaginae showed a better diagnostic effect, particularly with the combination of L. crispatus and G. vaginalis.