Clock synchronization is the key to ensuring the accuracy of time difference of arrival (TDOA) positioning systems. Currently, most TDOA systems adopt wireless clock synchronization methods, which necessitate frequent timing exchanges between master and slave base stations. Such wireless synchronization scheme suffers from susceptibility to interference, channel occupation issues, limited tag capacity and so on. Accordingly, a design for a TDOA positioning system utilizing UWB-based wired clock synchronization is proposed. This system has designed a differential remote transmission circuit for clock signal and synchronization signal. Particularly, the circuit for modulation and demodulation of the synchronous signal employs D flip-flop and NOR gate to ensure the phase alignment between the clock signal and the synchronization signal. Moreover, a clock synchronization calibration method designed to compensate for system errors, which confirms a synchronization accuracy of 0.5 ns with a maximum operational range of 100 m via standard Category 5e cables, superior to conventional wireless clock synchronization methods. In addition, a multi-tier cascaded clock synchronization architecture (Master→Submaster→Slave) was implemented to achieve high system scalability. Experimental verification in indoor environments demonstrates that the tag positioning derived from this TDOA positioning implementation yields an average R95 accuracy of approximately 9.75 cm. To sum up, the developed system enhances both precision and stability in UWB-based TDOA localization and reduce wireless channel occupancy of ultra-wideband TDOA positioning.