MiRNA (microRNA) plays an important role in plant growth, development and stress resistance by regulating its target genes. In this study, we used molecular biology and biochemistry methods to study the mechanism of cotton miR397LAC4 module involved in plant lignin biosynthesis and resistance to cotton bollworm. The results showed that: (1) cotton miR397 (ghrmiR397) regulated laccase gene (GhLAC4) expression after transcription, and GhLAC4 belongs to the blue copper oxidase family. It controls the synthesis of lignin and prevents cotton bollworm from invading cotton. (2) GUS reporter gene fusion expression and enzyme activity assay showed that ghrmiR397 directedly cleaved the target gene GhLAC4 after transcription and inhibited its expression. (3) Using VIGS (virus induced gene silencing) technology, miR397 was silenced and overexpressed in cotton. Cotton bollworm analysis showed that silencing ghrmiR397 expression would increase cotton resistance to cotton bollworm, but overexpression of ghrmiR397 would reduce cotton resistance. (4) The experimental analysis, histochemical staining and lignin content determination of selective and non selective cotton bollworm showed that silencing GhLAC4 would reduce the accumulation of lignin and increase the sensitivity of cotton to bollworm. These results show that ghrmiR397GhLAC4 module can fine tune cotton lignin synthesis to participate in the regulation of cotton insect resistance. At the same time, it also provides a new idea for cotton insect resistance breeding.