A new assay technique to distinguish between pure compounds and the isomeric mixtures has been suggested using single molecule (SM) fluorescence detection technique. Since the number of emission spots in a fluorophorespread film prepared from a genuine dye solution was determined by experimental condition, the deviation of spot numbers from the expected values could be considered to be an indication of lower purity of the sample solution. The lower limit of sample concentration for this assay was determined to be $5{ imes}10^{-10}$ M to show uniform number of expected spots within 10% uncertainties in our experimental condition. An individual fluorescence intensity distribution for a mixture of isomers having doubly different emissivities was simulated by adding distributions obtained from Cy3 and nile red (NR) independently. The result indicated that the mixture could be identified from the pure compounds through the difference in the number of Gaussian functions to fit the distribution. This new assay technique can be applied to the purity test for synthetic biofluorophores which are usually prepared in small quantities not enough for classical ensemble assays.