The timing synchronization system (TSS) in a radio-frequency–driven negative ion–based neutral beam injection system (NNBIS) is an important part of a negative ion–based neutral beam injection control system. In order to ensure the orderly conduct of experiments and the integrity of experimental data, the TSS needs to complete the corresponding timing synchronization function. There are two aspects to control of the NNBIS: controlling the synchronization of NNBIS subsystems within a certain precision and ensuring correct timing and amplitude output during the experiment. This paper presents the design and development of the TSS in the NNBIS, aiming at the characteristics of the IEEE 1588-2019, “IEEE Standard for a Precision Clock Synchronization Protocol for Networked Measurement and Control Systems,” clock synchronization protocol to achieve the demands of clock synchronization at a submicrosecond level, synchronously triggering other subsystems through the network trigger and hardware trigger. The TSS successfully implemented synchronization accuracy in less than 1 μs, and the network trigger is more efficient than the hardware trigger, which is about 80 μs faster. The TSS meets the requirements of timing synchronization accuracy of the NNBIS.