The rapid development of biomedical nanomaterials has placed higher demands on synthesis, analysis, and detection technologies. Currently, electrophoresis (EP) has become a powerful tool for studying biomedical nanomaterials, offering unique advantages in preparation, characterization, and delivery. Benefiting from the versatility of EP, researchers have successfully utilized various EP-based methods to develop nanomaterials, achieving significant advancements in biomedical applications. In this review, we provide a comprehensive overview of EP methods applied in biomedical nanomaterials, including EP methods for nanomaterial preparation, EP methods for nanomaterial characterization, and self-electrophoresis-driven nanomotor for drug delivery. We detail the design principles, working mechanisms, and recent progress in these fields. Moreover, we also discuss key challenges such as scalability, safety assessment, and integration with complementary techniques, and propose future research directions to further optimize EP methods for biomedical application. We hope this review will serve as a valuable reference for advancing the development of EP-based strategies in biomedical nanomaterials, ultimately contributing to innovative solutions in diagnostics, therapeutics, and personalized medicine.