〔Abstract〕 Objective To investigate the effects of probiotics on the functional metabolism of intestinal flora and clinical diseases in the neonatal intensive care unit( NICU) premature infants. Methods A total of 40 premature infants from the NICU were randomly divided into the probiotics group and the control group. Feces samples of two groups were collected on the 10 th day, and intestinal flora was detected and analyzed by 16 S rRNA high-throughput sequencing. And the differences in the incidence of clinical diseases were also compared between the two groups. Results Compared with the control group, the abundance of bifidobacterium and lactobacillus in intestinal flora of the probiotics group on the 10 th day was higher(P<0.05), while clostridioides, staphylococcus and cupriavidus bacteria were lower(P<0.05). In terms of functional analysis, the functions of the pentose phosphate pathway and nicotinate and nicotinamide metabolism in the probiotics group were higher than those in the control group(P<0.05), while the necroptosis was lower(P<0.05). In addition, the function gene expression levels of human pathogen sepsis(P<0.01), pneumonia, gastroenteritis and diarrhea(P<0.05) were also lower in the probiotics group. Moreover, the bilirubin and hemoglobin on the 10 th day(P<0.01) and the incidence of diarrhea and feeding intolerance(P<0.05) in the probiotics group were lower than those in the control group. And the incidence of sepsis and necrotizing enterocolitis( NEC) was also lower, but the difference was not significant. Conclusion This study shows that probiotics may have a significant effect on the metabolic function of intestinal flora and clinical disease in NICU premature infants, and explain the mechanism of probiotics to reduce the risk of clinical disease from the perspective of function.