Nerve root injury can occur in complex spine surgeries. Recording transcranial motor-evoked potentials (TcMEPs) has been the most popular method to monitor motor function during surgery. However, TcMEPs cannot detect single nerve root injury satisfactorily. Recently, multi-train stimulation (MTS) was demonstrated to effectively enhance TcMEPs. The aim of this study was to investigate the utility of TcMEPs elicited by MTS for intraoperative nerve root monitoring. TcMEPs were recorded from the quadriceps femoris (QF) and gastrocnemius (GC) muscles in the hindlimbs of 20 rats before and after transection of the nerve root at L6 (dominant root innervating the GC). For MTS, a multipulse (train) stimulus was delivered repeatedly at 5 Hz. The change ratio of the amplitude after transection of the nerve root was compared between MTS and conventional single-train stimulation (STS). The change in TcMEP amplitudes for QF after transection of the nerve root at L6 was 97.8 ± 12.2% with MTS and 100.1 ± 7.2% with STS (p = 0.496), whereas that for GC was 40.6 ± 11.5% with MTS and 64.8 ± 8.8% with STS (p < 0.001). MTS could improve the ability to detect isolated nerve root injury in intraoperative TcMEP monitoring.