A display substrate includes a first display region disposed on a substrate, wherein the first display region includes a plurality of light-emitting regions arranged in an array and a plurality of light-transmitting regions arranged in an array. The plurality of light-emitting regions and the plurality of light-transmitting regions are arranged alternately. Each of the light-emitting regions includes: a conductive layer disposed in the light-emitting region; and a cathode block disposed on the conductive layer. The first display region further includes connection portions that are disposed in the light-transmitting regions and located in a same layer and made of a same material as the conductive layers, and respectively configured to electrically connect cathode blocks of two adjacent light-emitting regions. Further provided is a manufacturing method of a display substrate.