In this work, a novel online three dimensional liquid chromatography (3D-LC) system was first developed by effectively coupling of preseparation and comprehensive 2D-LC using a stop-flow interface, aiming at improving the separation of complex samples. The sample was separated into two or several fractions through the first dimensional separation, and then each fraction was transferred in an orderly way into the following comprehensive 2D-LC part for further analysis. More optimal conditions could be operated in the second and third dimensions according to the properties of each fraction. Thus, the resolution of the 3D-LC system was substantially improved. Analysis of soybean extract was taken as a proof-of-principle to demonstrate the powerful separation of the established 3D-LC system. The amide column was selected as the first dimension column. Weakly polar metabolites (such as lipids, aglycones, etc.) and polar metabolites (such as glycosides, etc.) were separated into different fractions. Fluorophenyl and C18 columns were used in the second and third dimensions of the 3D-LC system for further separation, respectively. There were 83 flavonoids characterized in the soybean extract, including many difficult to separate isomers and low-abundance flavonoids; in total, they were nearly 30% more than those identified in the comparative comprehensive 2D-LC approach. In conclusion, this 3D-LC system is flexible in construction and applicable to complex sample analysis.