Accurate and rapid diagnosis of Acanthamoeba keratitis (AK) is difficult. Although the diagnostic procedure for AK has improved, further development and effective diagnostic tool utilization for AK need to continue. Chorismate mutase is a key regulatory enzyme involved in the shikimate pathway, a metabolic pathway absent in mammals but central for amino acid biosynthesis in bacteria, fungi, algae, and plants. In this study, we describe the identification and production of a polyclonal peptide antibody targeting chorismate mutase secreted by A. castellanii, which could be used for AK diagnosis. Western blot was performed using the protein lysates and conditioned media of the human corneal epithelial (HCE) cells, non-pathogenic Acanthamoeba, pathogenic Acanthamoeba, clinical isolate of Acanthamoeba spp., and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Polyclonal antibodies raised against A. castellanii chorismate mutase specifically interacted with lysates of Acanthamoeba origin and their culture media, while such interactions were not observed from other samples. Acanthamoeba-specificity of chorismate mutase was also confirmed using immunocytochemistry after co-culturing Acanthamoeba with HCE cells. Specific binding of the chorismate mutase antibody to Acanthamoeba was observed, which were absent in the case of HCE cells. These results indicate that the chorismate mutase antibody of Acanthamoeba may serve as a method for rapid and differential Acanthamoeba identification.