Pooled sample testing (PST) as a strategy for avoiding testing the majority of individual negative samples has been proposed for screening of diseases in low prevalence areas. There has been no standard guideline for PST in screening of Schistosoma japonicum infection of Yunnan, China. To document the optimum pool size with acceptable sensitivity of PST for screening of Schistosoma japonicum infection in this setting, an experimental pooling of each of 31 positive sera by IHA with various numbers of 24 negative sera was done. The results were used to create a statistical model which was subsequently used for simulation to predict sensitivity of the pooled serum tests in the population with varying prevalence and pool size. We found that to keep the sensitivity of PST above 90%, 1:05 should be the maximum dilution, that is, the optimum pool size should not be greater than 6. Antigen will have rather little interference if the prevalence of infection is low e.g. 1% or the antigen:antibody ratio is 1:100 or below. Pooled serum testing by IHA is an acceptable sensitive method for detecting antibody for Schistosoma japonicum infection in this area.