The construction of multifunctional nano-enzymes is a feasible strategy for fighting multi-drug resistant (MDR) bacterial biofilm-associated infections. Extracellular DNA (eDNA) is an important functional part of biofilm formation, including the initial adherence of bacteria to subsequent development and eventual maturation. A nano-enzyme platform of graphene oxide-based nitrilotriacetic acid-cerium(IV) composite (GO-NTA-Ce) against bacterial biofilm infection has been developed. When located at the site of bacteria-associated infection, GO-NTA-Ce could inhibit the biofilm formation and effectively disperse the formed biofilm by degrading the eDNA. In addition to Ce-mediated deoxyribonuclease (DNase)-like activity, near-infrared laser irradiation of GO-NTA-Ce could produce local hyperthermia to kill the bacteria that lost the protection by the biofilm matrix. In addition, graphene is also a new green broad-spectrum antimicrobial material that can exert its antimicrobial effects through physical damage and chemical damage. In short, our GO-NTA-Ce nano-enzyme platform is capable of effectively eradicating drug-resistant bacterial biofilm infections through the triple action of DNase-like enzyme properties, photothermal therapy, and graphene-based antimicrobial activity, and the nano-composite has excellent potential for the treatment of MDR bacterial biofilm infections.