We report a hydrothermal method to prepare reduced graphene oxide (rGO)-based hydrogel (GelrGONR), using neutral red (NR) to mediate the assembly of rGO sheets and tune the pore size of GelrGONR. A series of techniques including scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Raman spectroscopy and BET were employed to characterize the physico-chemical properties of GelrGONR. A large pore size of up to 20µm and interconnected porous structure of GelrGONR were obtained. GelrGONR was used as a support for immobilizing microbe (denoted as GelrGONR-M), which showed ~3.3 times more load mass of microbe than commonly used supports (i.e., activated carbon and carbon fiber felt) and 2.5 times higher biodegradation efficiency (BE) than carbon fiber felt. Further use of GelrGONR-M as a biocatalyst for establishing a BOD biosensor exhibits a linear range of 2-64mgOL(-1) and a detection limit 0.4mgOL(-1) for glucose-glutamic acid (GGA). Moreover, our proposed BOD detection strategy shows a long-term viability over one year and stability up to 2 months with a relative standard deviation of 2.1%. Our results demonstrated the great potential of employing GelrGONR as a microbe-immobilization support for biosensor development.