A light-emitting substrate includes; a base, an isolation portion disposed on the base and located in an isolation region located outside a light-emitting region, and a second insulating pattern located in the light-emitting region. The isolation portion includes a first conductive pattern, a second conductive pattern and a first insulating pattern that are sequentially stacked on the base; an orthogonal projection of the first conductive pattern on the base is located within an orthogonal projection of the second conductive pattern on the base; and a side face of the first conductive pattern proximate to the light-emitting region and a corresponding side face of the second conductive pattern proximate to the light-emitting region have a first gap therebetween. A side face of the second insulating pattern proximate to the first insulating pattern and a side face of the first insulating pattern proximate to the second insulating pattern have a second gap therebetween.