A transparent display substrate, a manufacturing method thereof, and a transparent display device are disclosed. The transparent display substrate includes a base substrate and a pixel defining layer defining a plurality of pixel regions on the base substrate; at least one of the pixel regions includes a light-transmitting portion and a light-emitting portion, and is provided with an OLED layered portion and an auxiliary electrode; the OLED layered portion includes a reflective anode, an organic light-emitting layer and a transmissive cathode; and the auxiliary electrode is located in the light-transmitting portion, disposed at a side of the transmissive cathode close to the base substrate, and connected to the transmissive cathode.