A method for the detection of bacterial pathogens in sepsis and bacterial meningitis with 16S rRNA gene- based real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) is developed. A total of 190 blood specimens and 5 cerebrospinal fluid specimens from neonates with suspected sepsis or bacterial meningitis were evaluated with 16S rRNA gene-based real-time FQ-PCR assay. The positive rate of the real-time FQ-PCR assay was significantly higher (25/195, 12.82%) than that of bacterial culture (15/195, 7.69%; P = .002). When bacterial culture was used as a control, the sensitivity of the real-time FQ-PCR was 100%, the specificity was 94.4%, and Youden's index was 0.944. This study suggests that 16S rRNA gene-based real-time FQ-PCR assay is an important and accurate method in the detection of bacterial pathogens of sepsis and bacterial meningitis and should have a promising usage in the diagnosis of sepsis and bacterial meningitis.