To analyze the clinical characteristics, pathogens, and inflammatory markers of premature infants with septicemia caused by nosocomial infection. Methods: The clinical data of 87 premature infants with septicemia caused by nosocomial infection from June 2016 to June 2022 were analyzed retrospectively. The clinical characteristics and inflammatory indicators were compared between the premature infants with Gram-negative bacteria infection and the premature infants with Gram-positive bacteria infection. Results: The incidence of septicemia of the premature infants caused by nosocomial infection was 1.07% (87/8146). In 59 (67.8%) premature infants with Gram-positive bacteria infection, staphylococcus epidermoides infection was the most of 33.9%, and in the 28 (32.2%) premature infants with Gram-negative bacteria infection, klebsiella pneumoniae was the most of 39.3%. The incidences of the early onset of septicemia, the serious complications, the leukopenia, the elevated C-reactive protein level, and the thrombocytopenia of the premature infants infected with Gram-negative bacteria were significantly higher than those of the premature infants infected with Gram-positive bacteria. The duration of fetus, the birth weight, the duration of mechanical ventilation, the antibiotic application rate, and the duration of PICC retention of the premature infants infected with Gram-negative bacteria were significantly more than those of the premature infants infected with Gram-positive bacteria. The hospitalization costs of the premature infants infected with Gram-negative bacteria was significantly higher than that of the premature infants infected with Gram-positive bacteria (P<0.05), and the mortality rate of the premature infants infected with Gram-negative bacteria was significantly higher than that of the premature infants infected with Gram-positive bacteria (P<0.05). Conclusion: The septicemia of the premature infants in nosocomial infection is mainly caused by Gram-positive bacteria. The premature infants with Gramnegative bacteria infection have more serious complications and worser prognosis. The decrease of white blood cells and platelets, and the increase of C-reactive protein level of the premature infants are helpful to identify the type of infection and treatment.