This paper reports a functionalized cationic poly(amidoamine) crosslinker synthesized by Michael addition of peptide mimetic agmatine to N,N′-bis(acryloyl)cystamine. A polyethylene glycol diacrylate (PEGDA) hybrid hydrogel was developed using a functionalized crosslinker with cell adhesion capability and tunable stiffness. This engineered hydrogel presents a 3-D in-growth of cells and the cryosection shows remarkable morphology of the gel. The tunable stiffness of this hydrogel can regulate the osteogenic differentiation of mesenchymal stem cells from RT-PCR. Both in vitro and in vivo tests demonstrate adjustable degradation rate and biocompatibility. The crosslinker and its hydrogels hold promising potential for tissue engineering applications.