Test was conducted on the samples from Yangtze River,Qinghuai River and Xuanwu Lake using enzyme substrate and multiple - tube fermentation techniques in parallel to look into the applicability of enzyme substrate technique to fecal contamination in surface water. The results indicated that in most cases the coliform values from enzyme substrate technique were lower than that from multiple -tube fermentation technique. However, there was no statistical discrepancy between them（ P 〉 0.05 ）. Compared with multiple -tube fermentation technique, enzyme substrate technique characterized with a shorter testing period and lesser secondary pollution, has a better specificity and a wide application.