Host-induced gene silencing (HIGS) has been extensively studied and applied in plant disease-resistant breeding in recent years; however, this technology first requires the establishment of small interfering RNAs (siRNA) targeting pathogen genes in host plants. Based on the concept that virus-induced gene silencing (VIGS) can rapidly generate siRNA in Nicotiana benthamiana, a fusion fragment capable of simultaneously silencing the GFP gene in transgenic N. benthamiana 16c (hereafter referred to as 16c-NB) and the CYP51 gene in Verticillium dahliae was constructed. This fusion fragment was inserted into the VIGS vector TRV2, enabling rapid confirmation of whether gene silencing had occurred by observing the disappearance of green fluorescence in 16c-NB, while also inducing the production of large quantities of siRNA targeting the V. dahliae CYP51 gene in 16c-NB. Results showed that the GFP gene in 16c-NB was silenced after transformation with the TRV2:GFP- CYP51 vector. Moreover, the wilt symptoms of 16c-NB transformed with TRV2:GFP- CYP51 were significantly less severe than those of control 16c-NB transformed with the TRV2:GFP vector after inoculation with V. dahliae. These findings indicated that siRNA targeting the V. dahliae CYP51 gene, generated in 16c-NB via VIGS, significantly enhanced the resistance of this plant species to V. dahliae. The results suggest that the TRV2:GFP-target gene system established based on VIGS provides an efficient tool for the rapid screening of HIGS target genes conferring resistance to V. dahliae.