Neonatal sepsis remains an important and common cause of morbidity and mortality among newborn infants, especially in developing countries. The aim of the present study was to determine whether serum trypsin levels and genotypes of cationic trypsinogen (PRSS1) gene could be served as markers for predicting neonatal sepsis. The serum trypsin levels and genotypes of PRSS1 were examined in both 50 infants with infection during neonatal period and 56 healthy neonates as controls. The infected infants were further subdivided into infants with sepsis group (n=18) and infected infants without sepsis (n=32). The genotype of PRSS1 was analyzed by direct sequencing, and the serum trypsin level was measured by immunoassay. It showed that the median value of serum trypsin was significantly higher in infected infants (31.90 ng/mL) than in controls (12.85 ng/mL; P=0.030). More importantly, sepsis subgroup (50.95 ng/mL) had significantly higher median serum trypsin than infected infants without sepsis subgroup (19.10 ng/mL) and controls (12.85 ng/mL) (P=0.015 and P=0.002, respectively). Additionally, the median serum trypsin levels were found significantly higher in infants who had T/T (37.90 ng/mL) genotype of PRSS1 compared with those who had C/T genotype (12.80 ng/mL; P=0.005). This study suggested that serum trypsin and rs10273639 C/T of PRSS1 were revealed to be novel markers for predicting neonatal sepsis.