Babesiosis is a tick-borne parasitic disease that poses a significant risk to both animal and human health. A comprehensive understanding of Babesia biology necessitates the application of advanced laboratory techniques. This review explores recent advancements in gene editing technologies of Babesia, emphasizing the foundational importance of in vitro culture systems. We highlight the historical challenges encountered in establishing effective in vitro culture and discuss the need for optimizing these methods to enhance gene editing efficiency. Here, we describe recent progress in Babesia transfection, different gene manipulation systems, and the applications of gene editing. This review aims to provide essential insights and technical guidance for future studies in Babesia genetics, highlighting the transformative potential of gene manipulation in combating this important parasitic disease.