Circulating microRNAs (miRNAs) are an emerging biomarker for sepsis patients. The purpose of this study was to identify novel miRNAs in the sera of sepsis patients and determine their prognostic value. Ninety-four serum samples were collected from sepsis patients within 24 h of intensive care unit admission. Solexa sequencing followed by bioinformatics analysis was used to predict novel miRNAs in survivors (n = 9) and nonsurvivors (n = 9). A total of 650 novel miRNAs were predicted by bioinformatics analysis after Solexa sequencing, and 41 novel miRNAs were validated in 10 survivors, 10 nonsurvivors, and 10 healthy controls by quantitative reverse transcriptase-polymerase chain reaction. Among these 41 miRNAs, 18 were present in both survivors and nonsurvivors, and nine were differentially expressed between the two groups. The expression levels of the nine miRNAs were determined by quantitative reverse transcriptase-polymerase chain reaction in 24 nonsurvivors and 32 survivors, and six were differentially expressed. Conjoint analysis of the six miRNAs and severity scores (Acute Physiology and Chronic Health Evaluation II score and Sequential Organ Failure Assessment score) showed that the area under the receiver operating characteristic curve for the predictive value of the six miRNAs was 0.969 (95% confidence interval, 0.930-1.000). When the cutoff point was set at 0.714, the six miRNAs and severity score provided a sensitivity of 100% and a specificity of 82.6%. In conclusion, 41 novel miRNAs were detectable in the sera of sepsis patients, and six of them might be related to sepsis outcome.