In this study, blood culture and PCR-microarray analysis were used to examine 172 cases of suspected septicemia. Primers and oligonucleotide probes, based on the sequences of bacterial 16SrRNA gene, were arrayed by imprinting on microarray slides. Blood specimens collected from 172 cases of suspected septicemia were cultured and then tested separately by PCR for the bacterial 16S rRNA. Of the 172 clinical cases, 17 cases tested positive by PCR. The number of positives identified by PCR (9.88%) was significantly higher than the number of positives identified by the blood culture (4.65%). When blood culture was used as control, the sensitivity of PCR was 100%, the specificity was 97.85%, and the index of accurate diagnosis was 0.979. When the 17 PCR positive specimens were further analyzed by hybridization against the microarrays, five were found to be probe positive for E. coli, four were positive for S. epidermidis, four were positive for CoNS, and two were positive for Bacillus and Propionibacterium, respectively. In the eight specimens showing positive results by both PCR and blood culture, the species determined by microarray analysis corresponded with the result obtained from blood culture. Detection of the bacterial 16SrRNA genes in clinical specimens by PCR and microarray analysis can be used to accurately diagnose neonatal sepsis. This method has a higher sensitivity and specificity than blood culture and can provide a rapid way for the etiological diagnosis of neonatal septicemia.