Objective To develop a method of simultaneous detection and Gram classification for pathogens causing sepsis with gram-specific probes based real-time PCR. Methods A pair of universal primers and a set of probes including Gram-positive probe and Gram-negative probe were designed based on the bacterial highly conserved region of 16SrRNA gene. With the gram-specific probes based real-time PCR, 35 clinical frequently-isolated strains including 17 gram-positive and 18 gram-negative bacteria were identified correctly with the corresponding gram probe. The blood samples from 512 cases of suspected septicemia, who were hospitalized in our neonatal ward and the NICU and developed clinical signs suggestive of infection, were tested with routine culture and bacterial gram-specific probes based real-time PCR separately. Results The detection limit of the gram-specific probes based real-time PCR assay was 10 CFU of the bacteria. The 35 isolates could be detected and classified correctly by gram-specific probes based real-time PCR. The PCR results were all negative for Cytomegalo virus, EB virus, hepatitis B virus, Cryptococcns neoformans, candida albican, human genomic DNA and negative control. The gram-specific probes based real-time PCR appeared to be quite specific. For 512 blood specimens from the patients with suspicious neonatal sepsis, the positive rate of the gram-specific probes based real-time PCR array was 8.20% (42/512,), which is significantly higher than that of blood culture (32/512, 6.25%) (χ2=8.10, P<0.01). When blood culture was used as a standard, the sensitivity of the gram-specific probes based real-time PCR was 100%. The specificity was 97.92% and the accuracy was 98.05%. Canclusions Cram-specific probes based real-time PCR with universal primers and gram-specific probes are developed. This study suggests that the bacterial gram-specific probes based real-time PCR are very useful for the rapid and accurate diagnosis of bacterial infection.