The aim of this study was to develop a rapid and sensitive diagnostic test for canine babesiosis caused by Babesia canis, based on the detection of parasite antigens in the blood of infected dogs using specific monoclonal antibodies. Three B. canis antigens (SPA, BcMSA1, Bc28.1) were used for in vitro immunization of murine B lymphocytes, which were subsequently fused with SP2/0-Ag14 myeloma cells. Hybridoma clones producing antigen-specific antibodies were selected, purified, and characterized using ELISA and Dot-Blot assays. The most promising clone was sequenced, and variable regions (VH and VL) were cloned into expression vectors and expressed in CHO-K1 cells. Recombinant antibodies were purified and incorporated into a prototype lateral flow immunoassay (LFIA). Diagnostic performance was evaluated using 100 blood samples from PCR-confirmed B. canis-infected dogs and 100 samples from healthy controls. Monoclonal antibodies specific to BcMSA1, Bc28.1, and SPA antigens were generated. Recombinant expression in CHO-K1 confirmed strong specificity to BcMSA1. In LFIA format, the assay demonstrated 90% accuracy with purified recombinant antigen and 70% accuracy with clinical samples (compared to PCR). The test showed complete specificity, with no false-positive results. The developed prototype of a rapid immunochromatographic test enables the detection of B. canis antigens in canine blood and may significantly improve clinical diagnosis of canine babesiosis. The test is rapid, simple, cost-effective, and suitable for routine veterinary practice.