A new graphene oxide (GO)-based hydrogel was synthesized through cross-linking of biofunctionalized graphene oxide nanosheets by di-alkyne polyethylene glycol as cross-linking agent. In this respect, nitrene chemistry as a convenient and straightforward protocol was developed for biofunctionalization of GO using an azido-starch as an eco-friendly, biodegradable and cost-effective material. In the next step, 1,3-dipolar cycloaddition chemistry, a green and highly efficient approach was utilized in cross-linking of functionalized GO by PEG through click reaction between remaining azido groups of starch on the surface of GO sheets and terminal alkyne groups of polyethylene glycol. Formation of aziridine and triazole rings during functionalization and cross-linking in this method could evidently improve biological activities of the obtained hydrogel compared to the conventional methods. The antibacterial activity of the new compounds was explored. The synthesized hydrogel showed antibacterial properties against Gram-positive and Gram-negative bacteria due to the presence of triazole rings. Also, the resulting hydrogel exhibited high dye removal efficiency and it can be utilized in water treatment effectively. The adsorption kinetics was analyzed through the effects of adsorption time and the dye concentration on the adsorption capacity. Kinetic data were accurately described by a pseudo-second-order model.