Abstract: The purpose of this study was to explore the gene expression profiles of follicles of different sizes during estrus in Murrah buffalo（Bubalus bubalis）by transcriptome sequencing technology, and to further understand their effects on related hormones during follicular development. Ovsynch method was used to synchronize estrus in Murrah buffalo. The size of follicles was detected by B-ultrasound and divided into three types : large, medium, and small（＞ 8 mm, 4 ～ 7 mm, 0 ～ 3 mm）. RNA was extracted from follicular fluid and sent to single cell transcriptome sequencing. The obtained data were compared between groups, and the differential transcripts were screened with |log₂fold change| ≥ 1 and P ＜ 0.05 as the standard. A total of 1 473 differentially expressed transcripts from the union of each group were selected for expression trend screening, in which 587 transcripts were in line with the transcriptional regulation law during bovine follicular development. Through GO and KEGG enrichment analysis, 33 pathways were screened, and each pathway was mainly related to intercellular interaction, steroid production and hormone production, cell differentiation and development, cell movement, metabolic regulation, physiological adaptability, and other processes. Through the analysis of the protein interaction network of the genes in the pathway, the key genes that may affect the hormone regulation during the development of buffalo follicles were further obtaind, such as BAMBI, FST, INHA, INHBA, INHBB, GATA6, and GFAP. In conclusion, these results provide a reference for further investigation of key genes and possible molecular regulatory mechanisms in the development of buffalo follicles.