The largest follicle at onset of predeviation(PDF1) and the second largest follicle at onset of deviation(ODF2) during bovine estrous cycle were collected by ultrasonic monitoring technology. The isolated granulosa cells(GCs) were subject to RNA library construction and Illumina 2000 sequencing. The resulted sequences were analyzed by Internet database RNA sequence search, differential expression screening, gene ontology and KEGG pathway analysis method, and genes sorting using Genecards to explore follicular development associated gene. The results showed that a total of 35 325 genes were identified after database gene search, among which were 15 645 significant expression genes with differentially expressed genes. The main functions of the 608 genes could be categorized into three groups: biological process(60.46%), cellular component(20.08%), molecular function(11.46%). KEGG pathway analysis showed that the 608 genes were involved in the regulation of bovine follicle development by 9 signal pathways(P<0.05). A total of 11 regulatory genes were obtained through genecards——SESN3, COL3A1, CCNE1, CAV1, CACNA1H, ITPR1, PIK3R3, MYC, PTGFR, CDK6 and GAPDH, which were closely related to bovine follicular development and directly involved in cell proliferation, differentiation and signal pathway regulation.