Sucrose phosphorylase, a hexosyltransferase, that is an important enzyme in starch and sucrose metabolisms, reversibly catalyzes the conversion of sucrose and orthophosphate to fructose and ${alpha}$-D-glucose-1-phosphate. A simple assay method for sucrose phosphorylase using 3,5-dinitrosalicylic acid (DNS) was developed. Its effectiveness was compared with that of a previously used NAD method. The results establish that the DNS method is comparable to the NAD method for the assay of sucrose phosphorylase. In particular, analysis of the enzyme activity level of sucrose phosphorylase (SPase) from Bifidobacterium longum SJ32 revealed that the DNS method is not only simple and accurate, but it also is a time-saving method for assaying sucrose phosphorylase activity. Most importantly, the DNS method is stable in broad pH ranges (pH 4-10), whereas the NAD method showed inaccurate profiles in the alkaline pH ranges (pH 8-10). Kinetic studies on SPase from B. longum SJ32 were performed using the simple DNS method developed in this study.