WRKY proteins, which form a transcription factor superfamily that responds to jasmonic acid (JA) signals, regulate various developmental processes and stress responses in plants, including Taraxacum kok-saghyz (TKS). TKS serves as an ideal model plant for studying rubber production and lays the foundation for a comprehensive understanding of JA-mediated regulation of natural rubber synthesis. In the present study, we screened and identified a valuable transcription factor, TkWRKY33, based on transcriptome data from TKS in response to JA. We investigated its role in the regulation of natural rubber synthesis within the JA signaling pathway and its function in response to drought stress. Through protein-protein interactions and transcriptional regulation analysis, we found that TkWRKY33 may regulate natural rubber synthesis through the JA-TkMPK3-TkWRKY33-(TkGGPS5/TkACAT8) cascade pathway, possibly by participating in JA-activated mitogen-activated protein kinase (MAPK) signaling. Overexpression of TkWRKY33 in tobacco, along with functional analysis of drought resistance and comparative analysis of natural rubber content after drought stress, revealed that TkWRKY33 not only enhances plant drought resistance by regulating the expression of genes related to reactive oxygen species (ROS) scavenging through the JA signaling pathway, but also has a close relationship with the signal transduction pathway mediated by the JA hormone in regulating natural rubber synthesis.The TkWRKY33 is recognized as a valuable transcription factor, which likely plays a role in regulating natural rubber biosynthesis through the JA-activated MAPK cascade signaling pathway JA-TkMPK3-TkWRKY33-(TkGGPS5/TkACAT8).