An on-line coupled comprehensive two-dimensional liquid chromatography (2D-LC) system was developed for the separation of triglycerides (TGs) in this work. Taking advantage of two-dimensional separation on the basis of two separation columns with different selectivities, where an silver-ion column and an octylsilica column were employed as the columns in the 1st and 2nd dimensions, respectively, all the TGs in perilla and tuna oils were well separated. Introducing the systematic qualitative analysis in gas chromatography (GC), it was possible to make the assignments for all the fatty acid compositions in the TGs that were fractionated after the comprehensive 2D-LC separations. In the subsequent GC analysis, 23 and 76 types of TGs were determined for perilla and tuna oils, respectively. The results clearly showed an excellent performance of the two-dimensional separation system for the separation of TGs in oils and fats, suggesting a future applications to the high performance separation of other types of fish oils with the on-line coupled comprehensive 2D-LC system developed in this work.