Rapid and accurate identification of specific sepsis pathogens is critical for patient treatment and disease control. This study aimed to establish a new application for the rapid identification of common pathogens in patients with suspected sepsis and evaluate its role in clinical application. A multiplex PCR assay was designed to simultaneously amplify specific conserved regions of nine common pathogenic microorganisms in sepsis, includingAcinetobacter baumannii, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumonia, andCandida albicans. The PCR products were analyzed by a membrane biochip. The analytical sensitivity of the assay was determined at a range of 5–100 copies/reaction for each standard strain, and the detection range was 20–200 cfu/reaction in a series dilution of simulated clinical samples at different concentrations. Out of the 179 clinical samples, the positive rate for pathogens detected by the membrane biochip assay and blood culture method was 20.11% (36/179) and 18.44% (33/179), respectively. However, by comparing the positive rate of the nine common pathogens we detected, the membrane biochip assay tended to be more sensitive than the blood culture method (20.11%vs15.64%). The clinical sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the membrane biochip assay were 92.9%, 93.2%, 72.2% and 98.6%, respectively. Generally, this multiplex PCR combined membrane biochip assay can be used to detect major sepsis pathogens, and is useful for early initiation of effective antimicrobial treatment, and is feasible for sepsis pathogens identification in routine clinical practice.