The invention relates to connection between chromatographic columns in a liquid-phase chromatographic separation system, in particular to a method for connecting the chromatographic columns in series in the liquid-phase chromatographic separation system, which comprises: firstly, correspondingly arranging a metal or PEEK joint on connecting ends of a main body chromatographic column and a joined chromatographic column respectively; and secondly, arranging a screen plate on end faces of the main body chromatographic column and the joined chromatographic column respectively, using a metal or PEEK two-way joint which is matched with joints to serially connect the main body chromatographic column and the joined chromatographic column into a whole, and locking the chromatographic columns and the two-way joint through corresponding internal and external threads on the two-way joint and the joints. The method has simple principle, conveniently increases the length of the columns through connection of the two-way joint, and improves the analysis ability of the chromatographic columns. Moreover, due to arrangement of the screen plates at both ends of the two-way joint, the method can effectively reduce the dead volume of column connection, increase the column effect of the columns in series connection, and prevent filler in the chromatographic columns from being flushed, and can be reassembled after disassembly.