A manufacturing method of an organic light emitting diode, OLED, substrate, wherein the manufacturing method comprises: a step S10 of providing a base substrate; a step S20 of forming an anode layer on the base substrate, wherein the anode layer comprises a first metal layer, a second metal layer, and a third metal layer stacked in sequence; wherein the step S20 of forming the anode layer on the base substrate comprising: a step S201 of depositing the first metal layer under a default cooling water flow rate condition of 4-6 standard mL/min; a step S202 of forming the second metal layer on a surface of the first metal layer via vapor deposition; a step S203 of depositing the third metal layer on a surface of the second metal layer under a default cooling water flow rate condition of 1-2 standard mL/min; and a step S204 of sequentially etching the third metal layer, the second metal layer and the first metal layer to obtain the anode layer; a step S30 of forming a pixel definition layer on the surface of the second metal layer, wherein the pixel definition layer comprises at least two pixel definition bodies, and the third metal layer is positioned between the adjacent pixel definition bodies; and a step S40 of forming a light-emitting layer on a surface of the third metal layer, wherein the light-emitting layer corresponds with the third metal layer, and the light-emitting layer is positioned between the adjacent pixel definition bodies; wherein film-forming conditions of the first metal layer, the second metal layer, and the third metal layer are different from each other. a step S10 of providing a base substrate; a step S20 of forming an anode layer on the base substrate, wherein the anode layer comprises a first metal layer, a second metal layer, and a third metal layer stacked in sequence; wherein the step S20 of forming the anode layer on the base substrate comprising: a step S201 of depositing the first metal layer under a default cooling water flow rate condition of 4-6 standard mL/min; a step S202 of forming the second metal layer on a surface of the first metal layer via vapor deposition; a step S203 of depositing the third metal layer on a surface of the second metal layer under a default cooling water flow rate condition of 1-2 standard mL/min; and a step S204 of sequentially etching the third metal layer, the second metal layer and the first metal layer to obtain the anode layer; a step S201 of depositing the first metal layer under a default cooling water flow rate condition of 4-6 standard mL/min; a step S202 of forming the second metal layer on a surface of the first metal layer via vapor deposition; a step S203 of depositing the third metal layer on a surface of the second metal layer under a default cooling water flow rate condition of 1-2 standard mL/min; and a step S204 of sequentially etching the third metal layer, the second metal layer and the first metal layer to obtain the anode layer; a step S30 of forming a pixel definition layer on the surface of the second metal layer, wherein the pixel definition layer comprises at least two pixel definition bodies, and the third metal layer is positioned between the adjacent pixel definition bodies; and a step S40 of forming a light-emitting layer on a surface of the third metal layer, wherein the light-emitting layer corresponds with the third metal layer, and the light-emitting layer is positioned between the adjacent pixel definition bodies; wherein film-forming conditions of the first metal layer, the second metal layer, and the third metal layer are different from each other.