A display substrate, a preparation method thereof, and a display apparatus are provide. The display substrate includes: a base substrate, an active layer disposed on the base substrate, a first gate insulating layer disposed on the active layer, a first conductive layer disposed on the first gate insulating layer, and a second conductive layer disposed on the first conductive layer and electrically connected with the first conductive layer; an orthographic projection of the first conductive layer on the base substrate does not overlap with an orthographic projection of the active layer on the base substrate; the second conductive layer includes gates; orthographic projections of the gates on the base substrate and the orthographic projection of the active layer on the base substrate have an overlap area; and the display substrate further includes: at least one insulating layer located between the first conductive layer and the gates.