A liquid ejection head substrate is manufactured by forming a wiring pattern on one surface of a substrate, forming an etching mask layer on the other surface of the substrate, forming a positioning reference mark on the etching mask layer by means of a laser, forming an opening pattern groove running through the etching mask layer and having a bottom in the inside of the silicon substrate, using the positioning reference mark, and forming a liquid supply port running through the silicon substrate by etching the silicon substrate from the opening pattern groove to the one surface by means of crystal anisotropic etching.