Genetic transformation in some plant species,including cotton(Gossypium hirsutum), is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing(VIGS) by double-stranded RNAs can serve as a reverse-genetics tool to determine gene function. However, knockdown levels vary greatly when using a tobacco rattle virus-based vector that carries different c DNA fragments of a gene. How to choose the optional target fragment for high interference efficiency is very challenging. Addressing this challenge requires increasing the efficacy of small interference RNA(si RNA) in target fragment. Here, we describe a method to assess VIGS efficiency by comparing the following parameters of si RNA in target sequence: the disruption energy of the target(DGdisruption), the differential stability of si RNA duplex ends(DSSE), and the internal stability at positions 9–14 of the si RNA antisense strand(AIS), which are calculated by Sfold program(http://sfold.wadsworth.org). We find that the si RNAs with low DGdisruption, high DSSE and high AIS have high activity and easily result in high VIGS efficiency by experimentally testing the actual knockdown levels of the four target genes, Gh PDS,Gh CLA1, Gh AOS1, and Gh CXE1 via choosing different target sequences for each gene. Therefore, the Sfold program can be used to analyze target sequences when carrying out VIGS design to increase gene-silencing effects in plants. Genetic transformation in some plant species, including cotton (Gossypium hirsutum), is hampered by laborious and time-consuming processes and often unachievable. Virus-induced gene silencing (VIGS) by double-stranded RNAs can serve as a reverse-genetics tool to determine However, knockdown levels vary greatly when using a tobacco rattle virus-based vector that carries different c DNA fragments of a gene. How to choose the optional target fragment for high interference efficiency is very challenging. Addressing this challenge requires increasing the efficacy of small interference RNA (si RNA) in target fragment. Here, we describe a method to assess VIGS efficiency by comparing the following parameters of si RNA in target sequence: the disruption energy of the target (DGdisruption), the differential stability of si RNA duplex ends (DSSE), and the internal stability at positions 9-14 of the si RNA antisense strand (AIS), which are calculated by Sfold program (http: //sfold.wadswo rth.org). We find that the si RNAs with low DGdisruption, high DSSE and high AIS have high activity and easily result in high VIGS efficiency by experimentally testing the actual knockdown levels of the four target genes, Gh PDS, Gh CLA1, Gh A Sfold program can be used to analyze target sequences when carrying out VIGS design to increase gene-silencing effects in plants.