The present invention discloses a pixel defining layer and a manufacturing method thereof, a display panel and a display device. The pixel defining layer comprises a first pixel defining layer and a second pixel defining layer stacked on the first pixel defining layer, wherein the first pixel defining layer has a plurality of first openings corresponding to light-emitting regions of respective sub-pixels in one-to-one correspondence, the second pixel defining layer has a plurality of second openings corresponding to the first openings in one-to-one correspondence, a cross section of the first opening is in a regular trapezoidal shape which is narrow at top and wide at bottom, and a cross section of the second opening is in an inverted trapezoidal shape which is wide at top and narrow at bottom. The present invention can effectively avoid the short between the anode and the cathode and the open of the cathode.