Objective To investigate the changes in intestinal flora in children with β-thalassemia major (β-TM) before and after hematopoietic stem cell transplantation and the influencing factors. Methods Children diagnosed with β-TM who underwent HSCT from November 2019 to June 2020 were enrolled as the research subjects. Fecal samples were collected during the pre-transplantation stage (group A), gut decontamination period (group B), conditioning period (group C), hematopoietic stem cell infusion period (group D), and post-transplantation cellular implantation period (group E). The fecal samples were sequenced using Illumina HiSeq sequencing system targeting the V4 region of the 16SrDNA gene. Results The study included 37 children with β-TM, including 27 males and 10 females with a median age of 7.5 (3.0-17.3) years. A total of 96 samples were analyzed, with 28 from group A, 17 from group B, 18 from group C, 21 from group D, and 12 from group E. The two most abundant phyla in groups A and B were Firmicutes and Bacteroidetes. In contrast, the two most abundant phyla in groups C, D, and E were Proteobacteria and Bacteroidetes. The differences in the abundances of Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Verrucomicrobia, and Fusobacteria among the five groups were statistically significant (P<0.05). The Chao was used to compare the alpha diversity of intestinal flora at different transplantation stages. The differences among the five groups were statistically significant (P<0.001). Similarly, the Shannon was used to predict alpha diversity of intestinal flora, and the differences among the five groups were also statistically significant (P<0.001). There were no statistically significant differences in the abundance and diversity of intestinal flora among the three groups treated with vancomycin, imipenem-cilastatin, and other broad-spectrum antibiotics (P>0.05). Conclusions The effect of hematopoietic stem cell transplantation on the composition of intestinal flora in children with β-TM was significant. The gut decontamination had little effect on the abundance of intestinal microbiota. The abundance and diversity of intestinal microbiota could not be restored in the early post-transplantation period. The selection of antibiotic types during HSCT cannot be optimized based on intestinal microbiota data.