Bioflocculant MBF-F2, MBF-F+, and MBF-F6, produced form culture of Rhizobium radiobacter F2, mixed culture of Rhizobium radiobacter F2 and Bacillus sphaeicus F6, and Bacillus sphaeicus F6, respectively, were investigated with regard to their production properties. The growth curves of the microorganisms were investigated. Effect of culture time on the flocculating activity and yield of bioflocculant were examined. Based on these experiments, 24 h was chosen as culture time in this study. In addition, three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy was applied to characterize the production of the bioflocculants under different culture time.