Ionic liquid (IL)-assisted sodium dodecyl sulfate polyacrylamide gel electrophoresis (ILs-SDS-PAGE) was presented to improve protein separation. ILs were employed during the preparation process of polyacrylamide gel, then the modified gel was used for commercial protein marker, binary bovine serum albumin/lysozyme (BSA/Lyz) and human serum separation. The influence of ionic liquid concentration, cation alkyl chain length, cation and anion types on proteins separation were investigated. The results showed that ILs played a role in improving some protein separation, and ILs-SDS-PAGE provided higher resolution and separation efficiency than ordinary SDS-PAGE for low and middle relative molecular mass proteins in human serum. In addition, the principle of ILs-SDS-PAGE was discussed and the comparison of ILs-SDS-PAGE with ordinary SDS-PAGE and Native PAGE was made.