A display substrate is disclosed, including pixel units defined by gate lines and data lines, and a first alignment layer. The first alignment layer includes first alignment grooves arranged in a first direction, the first direction forms a first intersection angle with a positive direction of a first side of each pixel unit, the first intersection angle is an obtuse angle, and the first intersection angle is smaller than an intersection angle between an orientation of liquid crystal molecule in a working state and the positive direction of the first side of each pixel unit. The direction of alignment grooves is decreased, so that the rotation angle of liquid crystal molecule is decreased, which reduces the responsive time of liquid crystal. A method for fabricating a display substrate, a display panel, and a display device are also disclosed.