The invention provides a hardware-in-loop fault injection system. The hardware-in-loop fault injection system includes: a fault injection matrix including a plurality of line channels and a plurality of row channels, wherein each line channel of the plurality of line channels and each row channel of the plurality of row channels are coupled via a short circuit relay, a signal simulation device is coupled to a device to be detected via the plurality of corresponding line channels as signal channels in the fault injection matrix, and each signal channel is provided with a cut-off relay; a power fault simulation unit which is coupled to the plurality of line channels in the fault injection matrix, wherein the plurality of line channels serve as power supply channels; and a fault injection monitoring unit which is used for controlling the cut-off relay of the fault injection matrix and on-off state of the short circuit relay, and monitoring on-off times of each relay, wherein the fault injection monitoring unit responds to a fault injection order, and the fault injection monitoring unit selects the relay for executing the fault injection order according to the on-off times of each relay.