Background: This study investigates the role of DNA topoisomerase II alpha (TOP2A) in retinoblastoma (RB), focusing on its involvement in epithelial-mesenchymal transition (EMT) and the potential of TOP2A inhibition as a therapeutic strategy. Methods: We analyzed TOP2A expression in RB tissues using public gene expression databases (GSE97508, GSE110811, and GSE172170) and conducted functional assays in human RB cell lines (Y79 and WERI-Rb-1) modified to knock down or overexpress TOP2A. Assessments included cell proliferation, migration, invasion, and EMT marker expression via RT-PCR and Western blot. Additionally, we evaluated the effects of TOP2A modulation in subcutaneous and liver metastasis mouse xenograft models. Results: TOP2A was significantly overexpressed in RB tissues (p < 0.0001). In vitro, TOP2A knockdown inhibited RB cell proliferation, migration, and invasion, and reversed EMT marker expression (p < 0.05), while TOP2A overexpression enhanced these oncogenic processes. In vivo, TOP2A knockdown or inhibition significantly reduced tumor growth and metastasis in both subcutaneous and liver metastasis models (p < 0.05). Combination therapy with TOP2A and EMT inhibitors further enhanced anti-tumor effects, significantly reducing tumor burden and metastatic lesions (p < 0.01). Conclusion: TOP2A is pivotal in RB pathogenesis and progression, primarily by regulating EMT. Its inhibition not only curtails RB cell proliferation and metastasis but also reverses EMT, underscoring its potential as a therapeutic target. This study lays the groundwork for further exploration of TOP2A-targeted therapies in RB.