A method of manufacturing a flow channel substrate, includes the steps of: forming two uneven shapes on a substrate in such a manner that the two uneven shapes follow a liquid flow channel at perimeters of a location where the liquid flow channel is to be formed and the two uneven shapes are configured to prevent a dissolvable resin forming a sacrificial layer from spreading during a heat treatment; forming, between the two uneven shapes on the substrate, the sacrificial layer which is made of the dissolvable resin and has a shape of the liquid flow channel; applying, by the heat treatment, a rounded shape to a corner section of the sacrificial layer on a side which is not in contact with the substrate; forming a coating resin layer on the substrate and the sacrificial layer; patterning the coating resin layer; and dissolving the dissolvable resin forming the sacrificial layer.