Multilayer ELISA (ML-ELISA) method for Entamoeba histolytica trophozoite coproantigen was developed for diagnosis of amoebiasis and its usefulness vis-a-vis microscopic examination analysed for paired specimens from 87 patients from Lucknow and Hyderabad, India. All the stool specimens positive for trophozoites were also found to be positive in ML-ELISA. Out of a total 32 samples positive for cysts in microscopy 21 (65.62%) gave positive reactions for coproantigen. Results on 26 samples drawn from otherwise healthy subjects with negative microscopical picture, 4 were positive for coproantigen. The results of the present study indicate that ML-ELISA test is suitable for early diagnosis of acute amoebic patients for timely treatment.