WRKY transcript factors(TFs) play crucial roles in plant response to biotic and abiotic stresses. However, how WRKY TFs response to aphid feeding are still poorly understood. Herein, NtWRKY28, a tobacco WRKY transcript factor gene induced by Myzus persicae feeding, was identified, and its regulatory roles were characterized in response to Myzus persicae feeding. The results showed that NtWRKY28 expression was induced by infestation of Myzus persicae, mechanical injury and MeJA treatment in tobacco plants. Overexpression of NtWRKY28 enhanced tobacco plant resistance to Myzus persicae, while silence of NtWRKY28 rendered tobacco plants more susceptible to infestation of Myzus persicae. Additionally, NtWRKY28 promoted the content of flavonoids and lignin through positively modulating the expression of genes involved in phenylpropanoid pathway, flavonoid and lignin biosynthesis. Our results not only provide new insights into the mechanism that WRKY TFs regulate tobacoo resistance to aphids, but also lay a theoretical foundation for breeding new tobacco varieties against aphids.