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Simple Summary: The laying hen industry plays an important role in the production value of the Chinese poultry industry, and the laying performance of hens is usually closely related to their follicle development. Studying the changes in the physicochemical state of the granulosa cells of laying hens during follicle development is helpful to better understand the order of development, which is of great significance in guiding laying hen breeding and production. In this study, transcriptome sequencing technology was used to reveal key regulatory genes in the follicular development of laying hens, and several potential target genes were screened. At the same time, we found that granulosa cell proliferation occurs mainly in the pre-hierarchical follicular period, whereas in the preovulatory period, granulosa cells complete their differentiation and begin to take on complex biological cell functions, including the synthesis of steroid hormones. Furthermore, as pre-hierarchical follicle granulosa cells progress to preovulatory follicle granulosa cells, the genes associated with steroid hormone synthesis increase dramatically, suggesting that steroid hormones play a critical role in follicle development. In this study, we aimed to better understand the difference between the functions of the two types of granulosa cells and sought to discover more key genes involved in follicle development and follicle selection. Herein, we separately collected pre-hierarchical follicle granulosa cells (PHGCs) and preovulatory follicle granulosa cells (POGCs) for RNA extraction; the transcriptomes of the two groups were compared via RNA-seq. A total of 5273 differentially expressed genes (DEGs) were identified between the PHGCs and POGCs; 2797 genes were up-regulated and 2476 were down-regulated in the PHGCs compared with the POGCs. A qPCR analysis confirmed that the expression patterns of 16 randomly selected DEGs were highly consistent with the RNA-seq results. In the POGCs, many of the genes with the most significant increase in expression were related to steroid hormone synthesis. In addition, the genes with the most significant decline in expression, including AMH and WT1, were related to the inhibition of steroid hormone synthesis. These results suggest that steroid hormones play a key role in follicle development. Furthermore, a Gene Ontology (GO) analysis revealed that these DEGs were mainly involved in the primary metabolic process, the carbohydrate metabolic process, the cellular process, ribosomes, the cytoplasm, and intracellular processes. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were mainly enriched in steroid biosynthesis, the cell cycle, ribosomes, the TGF-beta signaling pathway, focal adhesion, and so on. We also observed the morphology of the follicles at different developmental stages, and the results showed that the thickness of the granular layer of the small yellow follicles (SYFs) decreased significantly with further development. In addition, we also found that the thickness of the granulosa layer of hens over 300 days old was significantly lower than that of 200-day-old hens. In short, these data indicate that the tissue morphology and function of granulosa cells change throughout follicle development. [ABSTRACT FROM AUTHOR]