One-step purification and immobilization directly from crude extracts is a development trend to obtain high purity and performance target proteins due to the simple operation and high efficiency.Maltose binding protein (MBP)-tagged proteins usually need to be purified in biol. experimentsIt is very necessary to develop a low-cost, environmentally friendly and one-step purification and immobilization matrix.In this study, maltodextrin-modified hyperoxic graphene oxide (MHGO) was prepared using hyperoxic graphene oxide (HGO) and maltodextrin, which was characterized by SEM (SEM), (powder X-ray diffraction) PXRD and Fourier transform IR spectroscopy (FTIR).MHGO was then used as a one-step purification and immobilization matrix to treat the crude extract and obtain MlrA@MHGO with 84.2% purity of MlrA.The loading capacity of MHGO for MlrA was 379 ± 26 mg g-1 MHGO.Furthermore, the activity recovery of MlrA using the one-step strategy with MHGO as the matrix was found to be 26.5 times that of the two-step strategy.The Kcat value of MlrA@MHGO was 1.84 times that of free MlrA.Compared with free MlrA, MlrA@MHGO demonstrated enhanced stability, including half-life, thermal stability and pH stability, which were significantly improved than free MlrA.Based on these findings, we proposed a new paradigm for the purification and immobilization of MlrA using MHGO can be an effective matrix for the one-step purification and immobilization of MBP-tagged proteases.