Objective To search for genes related to the occurrence and development of retinoblastoma (RB). Methods In this study, firstly, we obtained 3 gene expression datasets from the Gene Expression Omnibus (GEO) database. After they were merged, the sva package in R software was applied to remove the batch effects of these three datasets. The differentially expressed genes (DEGs) were identified by limma package. ClusterProfiler package was used to analyze GO enrichment and KEGG pathway of DEGs. STRING database and Cytoscape software were used to construct the protein-protein interaction network (PPI). CytoHubba was applied to find the hub gene of PPI network. Weighted gene co-expression network analysis (WGCNA) was utilized to identify key modules associated with clinical information. The key genes of the key modules were further searched. Then, Y79, WERI-RB-1 and ARPE-19 cells were cultured in vitro, and the mRNA expression levels of five key genes were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A total of 20 RB tissue samples were collected. Immunohistochemistry was used to detect the expression of key proteins. Results A total of 1254 DEGs were identified, among which 422 were up-regulated and 832 were down-regulated. In GO analysis, DEGs were mainly related to protein heterodimerization activity, cation transmembrane transporter activity, and chromatin binding. In KEGG analysis, DEGs were mainly enriched in cell cycle, phototransduction, and DNA replication. A total of 79 hub genes in the PPI network were obtained. In the co-expression network, DEGS were divided into 11 co-expressed gene modules. According to Pearson correlation coefficient between each module and clinical traits, 5 important modules including blue, pink, turquoise, red and brown were identified, among which blue module had the highest correlation coefficient with age at diagnosis (r=0.65). After comprehensive analysis, we obtained 5 hub genes including structural maintenance of chromosome 4(smc4), minichromosome maintenance complex component 6 (mcm6), centromere protein K (cenpk), kinesin family member 15(kif15), protein regulator of cytokinesis 1 (prc1). The qRT-PCR results showed that the mRNA relative expression levels of prc1 and cenpk in Y79/WERI-RB-1 cells were higher than those in ARPE-19 (P<0.05). Immunohistochemistry results indicated that PRC1 protein was higher expressed in RB tissue samples (P<0.05). Conclusion smc4, mcm6, cenpk, kif15, and prc1 were identified as hub genes by bioinformatics analysis, and the increased expression of PRC1 protein in RB may play an important role in the occurrence and development of RB.