Babesia orientalis, a protozoan parasite transmitted by the tick Rhipicephalus haemaphysaloides, holds significant economic importance along the Yangtze River. Key factors in the host invasion process include rhoptry neck proteins (RON2, RON4, and RON5) and apical membrane antigen 1 (AMA1). However, the intricacies of the interaction between AMA1 and RONs remain incompletely elucidated in B. orientalis. To better understand these crucial invasion components, the RON4 gene of B. orientalis (BoRON4) was cloned and sequenced. RON4 is 3468 base pairs long, encodes 1155 amino acids, and has a predicted molecular weight of 130 kDa. Bioinformatics analysis revealed a unique region (amino acid residues 109-452) in BoRON4, which demonstrates higher sensitivity to epitope activity. The BoRON4 gene was strategically truncated, amplified, and cloned into the pGEX-6p-1 vector for fusion expression. We successfully used the mouse polyclonal antibody to identify native BoRON4 in B. orientalis lysates. Furthermore, the corresponding BoRON4 protein band was detected in the water buffalo serum infected with B. orientalis, while no such band was observed in the control. Additionally, I-TASSER and Discovery Studio software were used to predict the tertiary structures of BoRON4 and its ligands, CH-PKA and CH-complex. These ligands can serve as lead compounds for the development of anti-babesiosis drugs. In conclusion, BoRON4 emerges as a promising candidate antigen for distinguishing water buffalo infected with B. orientalis from their normal counterparts. This study positions BoRON4 as a potential diagnostic antigen for babesiosis in water buffalo, contributing valuable insights to the field of parasitology.