Aptamers are oligonucleotide molecules obtained by systematic evolution of ligands by exponential enrichment, which have a high affinity and specificity towards their target. Aptamers are applied in many fields, such as life science research, drug screening, target identification, and environmental monitoring. Affinity determination of an aptamer and its specificity are necessary for the research and development of the aptamer. Therefore, a novel affinity determination of aptamer, called ssDNA concentration method, was developed to study the specificity and affinity constant of an aptamer against . The affinity and affinity constant of the aptamer were determined by measuring the ssDNA concentration of the aptamer binding to the V. harveyi detected by the ssDNA concentration method, was 15.2 times higher than that of the non-target bacteria. The affinity and specificity of the aptamer were also verified by fluorescence microscopy. There are two main methods of fluorescence microscopy, the direct observation method and the fluorescence blocking method. The direct observation method is observing the binding of aptamer and target bacteria under fluorescent microscope after bacteria were incubated with fluorescent labeled aptamer. The results verified by the direct observation method under fluorescence microscopy showed that the target bacteria emitted significant yellow-red fluorescence after binding with the FAM-labeled aptamer, but little fluorescence was found in other four kinds of non-target bacteria (Escherichia coli), proving that the bacteria have no spontaneous fluorescence. Furthermore, the fluorescence blocking method involves incubating bacteria with no fluorescent label aptamer first, followed by incubating with fluorescently labeled aptamer, and observing whether the fluorescence is blocked under the microscope. We found that all bacteria including the target bacteria had no significant fluorescence. These results of the fluorescence blocking method proved that the aptamer had an excellent affinity and specificity towards the target bacteria , which also verified the determination of the ssDNA concentration method. The affinity constant () of the aptamer measured by the ssDNA concentration method is equal to 33.70±7.83 nmol/L, and its corresponding fitting coefficient is equal to 0.960, which showed that the ssDNA concentration method is accurate, reliable, and feasible in the determination of affinity and affinity constant of an aptamer. The ssDNA concentration method used in this study is practical due to the simple equipment, short measurement time, and convenient operation.