Objective　To investigate the characteristics of intestinal flora in breast-fed neonates with severe hyperbilirubinemia (breast milk jaundice, BMJ). Methods　Six breast-fed children with severe hyperbilirubinemia and six healthy breast-fed children from Shanghai Children's Medical Center affiliated to Shanghai Jiaotong University School of Medicine during January 2018 to June 2018 were enrolled. Stool samples were collected, and 16S rDNA sequencing was used to study the intestinal flora of 6 severe hyperbilirubinemia neonates (BMJ group) and 6 normal breastfeeding newborns (control group). Results　There was no significant difference in the diversity of intestinal flora between BMJ group and control group (P>0.05). At the genus level, the abundance of Escherichia was higher in BMJ group than that in control group (P＜0.05); LEfSe analysis showed Staphylococcaceae, Staphylococcus, Klebsiella, and Bacillales were higher in healthy control group. Conclusion　Although there is no significant difference in the diversity index of the intestinal flora between neonates with severe hyperbilirubinemia and jaundice-free breastfeeding infants, some bacteria have significant differences in the structure of the flora.