The samples were taken from the shallow urban lakes(Lake Yuehu) in Wuhan city to optimize the determination of dehydrogenase activity (DHA) in sediment, which gave the following suitable conditions: the sediment sterilized by formaldehyde was used as the control, the concentration of electron acceptor (2,3,5-triphenyl-2H-tetrazolium chloride) was 0.4%, the sediment amount used was 0.5g, pH value was 7.5 and the reaction time was 3h. Under these conditions, the DHA assayed was 3-5 times higher, and its positive relation to the content of organic matter in sediment was more significant, compared to those determined by the unmodified method. The data based on the 23 sites from 3 basins of Lake Donghu demonstrated the feasibility of the method. In the same time, the ecological significance of DHA was further shown in terms of the measurements of microbial activities and organic matter status in lake sediment