The invention provides a chromatographic separation and benzene ring number determination method for water-soluble organic matters. The method comprises the steps of preparing a water-soluble organic solution, adding the prepared water-soluble organic solution into a chromatographic column, subjecting the water-soluble organic solution to concentration/gradient elution through a fixed organic phase, detecting components obtained through the elution process by means of a fluorescence detector, and determining the number of benzene ring functional groups in each of different components according to the position of a maximum fluorescence peak in the emission spectrum of the separated components. The chromatographic separation and benzene ring number determination method has the advantages of small demanded sample quantity, good separation effect and accurate determination of benzene ring functional groups.