The invention relates to a biological graphene oxide antibacterial liquid and a preparation method thereof, and belongs to the technical field of preparation of graphene antibacterial liquids. The technical problems that in an existing graphene antibacterial liquid preparing process, when graphene is added into a dispersing agent or the graphene is added into an organic surface treating agent, exogenous substances are introduced, and secondary pollution is caused during sterilization are solved. The method includes the steps that S1, graphene oxide prepared through an oxidation reduction method is selected, water serves as a solvent, and a graphene oxide dispersion stripping solution is prepared through ultrasonic and centrifugal treatment; S2, the graphene oxide dispersion stripping solution prepared in S1 is subjected to dialysis treatment, the graphene dispersion liquid obtained after dialysis treatment is filtered through a sterile microporous filter membrane with the pore diameterof 0.22-0.45 micron, and the graphene oxide antibacterial liquid is obtained. According to the method, water serves as the solvent, other chemical reagents are avoided, the graphene oxide antibacterial liquid is free of toxins and environmentally friendly, and the graphene oxide antibacterial liquid can be used for spectrum antibacterial application and achieve a function of inhibiting bacteria.