The present disclosure provides a detection substrate, a manufacturing method thereof and a ray detector. The detection substrate includes: a base substrate; a plurality of independent first electrodes arranged on the base substrate on the same layer; a photoelectric conversion layer arranged on a whole face of sides, facing away from the base substrate, of the plurality of first electrodes; a ray absorption layer arranged on a side, facing away from the plurality of first electrodes, of the photoelectric conversion layer, wherein an orthographic projection of the ray absorption layer on the base substrate is overlapped with an orthographic projection of gaps between the first electrodes on the base substrate; and a second electrode arranged on a whole face of a side, facing away from the plurality of first electrodes, of the photoelectric conversion layer.