This study aimed to explore the underlying mechanism of relapsed acute lymphoblastic leukemia (ALL).Datasets of GSE28460 and GSE18497 were downloaded from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) between diagnostic and relapsed ALL samples were identified using Limma package in R, and a Venn diagram was drawn. Next, functional enrichment analyses of co-regulated DEGs were performed. Based on the String database, protein-protein interaction network and module analyses were also conducted. Moreover, transcription factors and miRNAs targeting co-regulated DEGs were predicted using the WebGestalt online tool.A total of 71 co-regulated DEGs were identified, including 56 co-upregulated genes and 15 co-downregulated genes. Functional enrichment analyses showed that upregulated DEGs were significantly enriched in the cell cycle, and DNA replication, and repair related pathways. POLD1, MCM2, and PLK4 were hub proteins in both protein-protein interaction network and module, and might be potential targets of E2F. Additionally, POLD1 and MCM2 were found to be regulated by miR-520H via E2F1.High expression of POLD1, MCM2, and PLK4 might play positive roles in the recurrence of ALL, and could serve as potential therapeutic targets for the treatment of relapsed ALL.