To provide a probe unit which can be aligned with respect to an electronic device being an object for inspection with high accuracy, and to provide its manufacturing method.

The probe unit includes a substrate having through holes, conducting wires which are formed on a first surface of the substrate by the use of lithography and come in contact with the electrodes of a specimen, and inspection alignment marks at least portions of the sidewalls of which are formed in the through holes by the use of the lithography into the shape of columns separated from the sidewalls of the through holes, and are aligned with respect to the specimen in a state of being observed along with the specimen from the second surface side of the substrate being the rear face of the first surface.