Quantitative real time PCR (qPCR)is used for pertussis diagnosis. The positive rate of qPCR is generally much higher than that of bacterial culture, which may cause confusion. The current study utilized the 16S ribosomal RNA (16S rRNA) sequencing to assess the correlation between conventional culture and qPCR and to explore the value of 16S rRNA in diagnosing pertussis. Nasopharyngeal swabs, collected from 102 children meeting clinical diagnostic criteria for pertussis, were subjected to Bordetella pertussis culture and qPCR. Bioinformatic microbiota analysis was based on 16S rRNA V3-V4 gene sequencing. Among 102 samples, 14 (13.7%) were culture-positive for Bordetella pertussis, while 61 (59.8%) were qPCR positive. Genus Bordetella was identified in 68 (66.7%) samples via 16S rRNA sequencing. When the relative abundance of Bordetella genus exceeded 0.70%, both qPCR and culture results were positive. Samples with a relative abundance of less than 0.20% exhibited positive qPCR and negative culture results. Samples with a low Bordetella abundance are the key factors underlying poor correlation between culture and qPCR results in laboratory tests.