L-Theanine contributes to the function and taste of tea. Ethylamine is a limiting factor that distinguishes L-theanine accumulation between tea and other plants. It has long been believed that ethylamine is derived from the L-alanine in tea. In this study, it was found that L-alanine in tea tree root cells is mainly located in vacuoles and mitochondria, while alanine decarboxylase (CsADC) in tea is located in the cytoplasm using non-water separation techniques. Although CsADC is capable of catalyzing the decarboxylation of L-alanine to produce ethylamine in vitro, it may not provide the same enzymatic activity in tea plants. Tracing of stable isotope-labeled precursors in tea plants found that L-alanine is not a direct precursor to ethylamine, but a precursor to L-glutamate, which is involved in L-theanine biosynthesis in tea. The epidermis layer from the root tissue is the main distribution site of ethylamine. In summary, in tea plants, L-alanine is converted to L-theanine by L-glutamic acid instead of ethylamine.