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<i>Monochamus alternatus</i> Hope (Coleoptera: Cerambycidae), an invasive beetle that has caused billions of dollars in economic losses, is a serious pest of <i>Pinus massoniana</i> in many Asian countries. Clarifying the eye pigment gene and its knockdown phenotype of <i>M</i>. <i>alternatus</i> can provide functional gene identification and a marker for screening of gene editing, as well as help develop new control ideas. In this study, we first screened the transcriptome and found one homologous gene of tryptophan 2,3-dioxygenase (<i>TDO</i>) and one of kynurenine-3-monooxygenase (<i>KMO</i>). By measuring the expression levels of <i>TDO</i> and <i>KMO</i> in different developmental periods, it was indicated that <i>TDO</i> and <i>KMO</i> were expressed in various stages of <i>M. alternatus.</i> The gene expression of <i>MaKMO</i> was higher than <i>MaTDO</i>, showing high expression after pupation and decreasing at the beginning of eclosion. <i>MaTDO</i> and <i>MaKMO</i> were knocked down using RNA interference technology in different periods of expression, and the temporal expression changes were obtained using RT-qPCR technology. The results showed that the expressions of <i>MaTDO</i> and <i>MaKMO</i> were significantly inhibited by the injection of dsRNA; the expressions of <i>MaTDO</i> at 48 h, 96 h and after pupation were 21.9%, 32.3% and 59.2%, respectively, meanwhile, those of <i>KMO</i> were 23.4%, 25.0% and 69.7%, respectively. There was a significant change in eye color, and the beetles were able to pupate normally without their activity being affected. Therefore, both <i>MaTDO</i> and <i>MaKMO</i> can be used as tag genes for <i>M. alternatus.</i> A dominant marker system based on eye color can be developed for the genetic manipulation and control of <i>M. alternatus.</i>