Abstract: Objective To investigate the clinical value of 16S rRNA gene in rapid diagnosis of neonatal sepsis. Methods The DNA of 37 strains of common pathogenic bacteria was amplified by polymerase chain reaction(PCR) with a pair of universal primers synthesized based on the highly conservative sequence of bacterial 16S rRNA gene,and the specificity and sensitivity of the PCR were tested.Blood samples from 106 cases of suspected sepsis neonates were collected.The DNA,blood culture,non-specific inflammatory index and procalitonin(PCT)of these neonates were tested and compared with 20 non-sepsis cases with x2-test. Results The 371 bp DNA fragment was amplified successfully from all bacteria strains,which had no cross-reactions with human DNA or virus DNA.Of 106 neonates with suspected sepsis,15 were positive for blood culture and 36 for PCR.The sensitivity,specificity and diagnosis index of PCR were 82.9%,96.9% and 179.85 respectively,which were better than those of blood culture and non-specific criteria.Compared with PCT,the specificity of PCR was significantly higher in perinatal neonates. Conclusions 16S rRNA gene amplified by PCR could be used to early detect the pathogenic bacteria in the specimens.It has higher sensitivity and specificity in diagnosis of neonatal sepsis as well as high value in distinguishing it from localized infection and non-bacterial infection.