The use of Strongyloides ratti as heterologous antigen for serodiagnosis of strongyloidiasis is preferable to Strongyloides from humans due to the ease and safety of antigen preparation. In Southeast Asia where Opisthorchis viverrini coexists with Strongyloides stercoralis, there has been no report in using S. ratti for serodiagnosis of S. stercoralis. In this study, performance of an enzyme-linked immunosorbent assay (ELISA) based on S. ratti was compared with that based on S. stercoralis for diagnosis of strongyloidiasis in areas where O. viverrini is co-endemic in Thailand. Of the 107 individuals, 50 (46.7 %) were positive for S. stercoralis by agar culture method and by ELISA; 82 (76.6 %) and 81 (75.7 %) were seropositive using S. ratti and S. stercoralis antigens, respectively. The levels of parasite-specific IgG to S. ratti and S. stercoralis antigen were significantly proportionally correlated (P < 0.001). Mixed infections with O. viverrini have little effect on diagnosis of strongyloidiasis. Of 42 subjects who were infected with other parasites, there were no cross-reaction with Angiostrongylus cantonensis, Taenia spp., hookworms, Paragonimus spp., Clonorchis sinensis, Ascaris lumbricoides except for Fasciola spp. (1 of 5), and Opisthorchis viverrini (5 of 20). In spite of cross-reactivities, the results suggest that the S. ratti antigen provides an useful option for diagnosis of strongyloidiasis in an endemic area of opisthorchiasis with high sensitivity comparable to the S. stercoralis antigen and provide a basis for effective control strategies for strongyloidiasis.