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Conjugates of semiconductor quantum dots (QDs) and organic dyes have been receiving attention as fluorescence biological sensing materials. In designing such sensors, a most important parameter is the number of organic-dye molecules that conjugate to a QD. If a precise separation method was developed, it might be possible to control conjugation without knowing the exact number of conjugated dye molecules per QD. In this study, the difference in linear velocities in a gel filtration column between CdSe/ZnS QDs and 5-(and 6)-carboxynaphthofluorescein succinimidyl ester is used. The velocities differ because the hydrophilicity of CdSe/ZnS QDs is much higher than that of the organic dye; hence, CdSe/ZnS-organic-dye conjugation can be controlled by changing the fraction number. Furthermore, the concentrations of CdSe/ZnS QDs and organic dye in fractionated solutions can be determined by measuring fluorescence spectra, and we demonstrate a fluorescence-type pH sensor based on the conjugate, which has a pH-sensitivity range from 7.5 -9.5.