To measure the depth of the through silicon vias on 300 mm silicon wafers, a measuring machine was developed. Based on the preceding research in a laboratory environment, the machine was designed and built by modifying the optical probe for reducing the mass, combining a visible optical microscope to monitor the location of the measuring points, and constructing the metrology frame for large silicon wafers. The depths of the three different-sized through silicon vias were measured repeatedly to estimate the repeatability. Moreover, comparative measurement was carried out to verify the measured depth values. The total measurement time was also estimated by measuring 110 through silicon vias at different locations. According to the measurement results, the measurement performance satisfied the technical requirements of the industry in terms of repeatability, accuracy, and measurement time.