A hydrogel is a solid form of polymer network absorbed in a substantial amount of aqueous solution. In electrophoresis, hydrogels play versatile roles including as support media, sieving matrixes, affinity scaffolds, and compositions of molecularly imprinting polymers. Recently, the study of hydrogels has been advancing with unprecedented speed, and the application of hydrogels in separation science has brought new opportunities and possible breakthroughs. A good understanding about the roles and effects of the material is essential for hydrogel applications. This review summarizes the hydrogels that has been described in various modes of electrophoretic separations, including isoelectric focusing gel electrophoresis (IEFGE), isotachophoresis (ITP), gel electrophoresis and affinity gel electrophoresis (AGE). As microchip electrophoresis (ME) is one of the future trends in electrophoresis, thought provoking studies related to hydrogels in ME are also introduced. Novel hydrogels and methods that improve separation performance, facilitate the experimental operation process, allow for rapid analysis, and promote the integration to microfluidic devices are highlighted.