According to a manufacturing method of an optical waveguide substrate including a core (12) and clads (11) (13) and provided with an optical axis conversion mirror (14) in the core and an alignment recess for the optical axis conversion mirror with respect to a light receiving and emitting element, the recess is obtained by obtaining an outline of the core by synthesizing at least an image captured by focusing a microscope (20) to a highest position (14a) of the core in an optical axis conversion mirror portion and an image captured by focusing the microscope to a lowest position (14d), and by determining a position of the alignment recess in reference to a center of gravity of the outline, and according to an optical waveguide substrate obtained by this manufacturing method, it becomes possible to provide an optical waveguide substrate in which the optical axis conversion mirror in the optical waveguide substrate and the light receiving and emitting element are aligned with respect to each other at an extremely high degree of accuracy and a manufacturing method thereof.