•This study established a transient transformation system of tea plant leaves using antisense oligodeoxynucleotide (asODN) for gene suppression, by optimizing the concentration of AsDON and the number of days of injection.•The reliability of the gene suppression transient transformation system was verified by the gene expression level and metabolic level in theanine/anthocyanin biosynthesis pathways.•This study established a transient transformation system of tea plant leaves using agrobacterium for gene over-expression, by optimizing the injection solution concentrations, and sampling time post injection.•The reliability of the gene over-expression transient transformation system was verified by GFP fluorescence signal and protein level. The system was further proved by the gene expression level and metabolic level in theanine/anthocyanin biosynthesis pathways.•The over-expression transient transformation system was proved can be used to study the subcellular localization of tea plant proteins in vivo. Tea, the most consumed non-alcohol beverage in the world, has many specialized secondary metabolites, and functional identification of their related genes is very important for the improvement of tea quality. However, a transient transformation system, which is considered as a valuable tool for gene function and protein subcellular localization, has not been established in tea plants. Here, we report a rapid and efficient system for transient transformation of tea plant leaves using antisense oligodeoxynucleotide (asODN) for gene suppression and Agrobacterium for gene over-expression. Specifically, we established the transient transformation system by analyzing different varieties, injection solution concentrations, and sampling time post injection, followed by green fluorescent protein (GFP) fluorescence signal and western blot analysis to confirm the reliability of the over-expression transient transformation system. Additionally, the suppression and over-expression effects of important genes relate to secondary metabolism were evaluated at gene expression and metabolic levels. Finally, a transcription factor was transiently over-expressed to determine its subcellular location by protoplasts successfully in the tea plant leaves. Our data indicate that this system is reliable and effective for studying gene function and protein localization in tea plant leaves, providing new insights into tea plant biology research.