The Pacific white shrimp (Penaeus vannamei), one of the world's most economically important aquatic species, is highly susceptible to Ecytonucleospora hepatopenaei (EHP), a pathogen that infects the hepatopancreas and causes hepatopancreatic microsporidiosis (HPM), leading to stunted growth and substantial economic losses in shrimp farming. Currently, no effective treatments for EHP exist, making rapid on-site detection and preventive measures essential for disease control. While nucleic acid-based detection methods are commonly employed, they require specialized equipment, controlled environments, and trained personnel, which increase costs. To address this limitation, we developed a colloidal gold immunochromatographic assay (GICA) strip for rapid on-site detection of EHP in shrimp farms. Using LC-MS/MS, 15 high-abundance EHP proteins were identified, with EhSWP3 ranked highest and selected as the optimal antigen detection target. Recombinant EhSWP3 was used to immunize mice, resulting in the development of monoclonal antibodies. The optimal capture and labeled antibody combination (1B6, 3A6) was identified and incorporated into the GICA strip. Testing with common shrimp pathogens and various microsporidia samples demonstrated the high specificity of the EHP test strip. The strip exhibited a sensitivity of 1.81 × 10