The invention provides a method for patterning a graphene layer and a method for manufacturing a display substrate. The method for patterning a graphene layer comprises: forming an isolation layer on a graphene layer; forming a photoresist layer on the isolation layer; patterning the photoresist layer; etching the isolation layer according to the patterned photoresist layer to form a patterned isolation layer; etching the graphene layer according to the patterned photoresist layer to form a patterned graphene layer; and removing the patterned isolation layer. In the method of the invention, the unfavorable condition of the prior art may be avoided that a graphene film sloughs off or a photoresist remains on a graphene film when a photoresist material is peeled off, and the product yield can be improved in the case that the production cost is controlled.