A microchannel chip has been used to fractionate selected segments from an electrophoretic flow of separated fragments. A sample, which covers the size from 35 to 670 bp, was initially separated using an 8.8-cm-long channel at the electric field strength of 100 V/cm. The target fragment of 318 bp was selected and extracted from the separation channel. High-resolution fractionation was achieved by introducing new procedures for blocking, extraction, and segment transfer. Fractionation quality with and without blocking were compared using a 310 Genetic Analyzer (Applied Biosystems). The results show that no contamination was found in the sample, which was fractionated with blocking; however, a contamination by short segments was found in the sample, which was fractionated without blocking. Furthermore, fractionation by the chip was found to be of higher fidelity than that by the polyacrylamide slab gel, which displayed a small overlapped peak after the target peak. Compared with the traditional method, our chips enable faster and high-fidelity fractionation, thus providing a new toot for bioanalysis and other applications.