Objective To investigate the pathogenic distribution and clinical characteristics of premature infants with septicemia in intesive care unit(ICU). Methods 155 premature infants with septicemia confirmed by laboratory positive blood culture were retrospectively analyzed, these infants were admitted in ICU of a hospital from January 2016 to December 2018, clinical characteristics of septicemia in premature infants with different pathogens were compared. Results Among 155 premature infants with septicemia, 76 cases (49.03%) were in Gram-negative bacillus group (G^- group), mainly Klebsiella pneumoniae septicemia; 42 cases (27.10%) were in Gram-positive coccus group (G⁺ group), mainly Staphylococcus epidermidis septicemia; 37 cases (23.87%) were in fungus group (fungus group), mainly Candida parapsilosis septicemia and Candida albicans septicemia. The gestational age and birth weight of premature infants in G^- group and fungus group were less than G⁺ group; disease onset age of premature infants in fungus group was older than G^- group and G⁺ group, proportion of deep vein catheterization was higher than G^- group and G⁺ group; platelet count of premature infants in fungus group was lower than G^- group and G⁺ group; C-reactive protein (CRP) of G^- group was higher than G⁺ group and fungus group, difference were all statistically significant (all P<0.05). The recovery rate and mortality of 155 premature infants were 94.84% and 5.16% respectively. Mortality of premature infants in G^- group was higher than G⁺ group and fungus group, treatment days of fungus group were longer than G^- group and G⁺ group,differences were all statistically significant (all P<0.05). Conclusion The main pathogens of septicemia in premature infants are G^- bacteria, clinical manifestations, laboratory markers and prognosis of septicemia children in G^- group, G⁺ group and fungus group are different.