Conventional polyacrylamide hydrogels prepared from the free radical polymerization between acrylamide and N,N¢-methylenebisacrylamide (NMBA) have been frequently used in the biochemical technique like the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to resolve protein mixtures. In this study, we have prepared an alternative polyacrylamide hydrogel from the cross-linking of acrylamide and N,N¢-bisacrylylcystamine (BACy). In addition, we have compared the BACy-based hydrogel with the NMBA-based polyacrylamide hydrogel for their physical properties such as swelling ratio, shear modulus, crosslink density and morphology. Moreover, we further determined whether BACy-based polyacrylamide hydrogel could be applied to SDS-PAGE and proteomics research. The results showed that this type of hydrogel is capable of separating proteins and facilitates further in-gel protein digestion and the following protein identifications by mass spectrometry. In summary, our study provides a basis for the putative application of BACy-based hydrogels.