L-Theanine is the most popular nonprotein amino acid contained in tea leaves. It is one of the umami components of green tea, contributing to the unique flavor of tea. Because of its various health functions, L-theanine has been commercially developed as a valuable ingredient easily used for various applications in food and pharmaceutical industries. Nowadays, L-theanine is mass-produced by plant extraction, chemical synthesis, or enzymatic transformation in factories. This review embodies the available up to date information on the L-theanine synthesis metabolism in the tea plant as well as approaches to produce it, placing emphasis on the biotransformation of L-theanine. It also gives insight into the challenges of L-theanine production on a large scale, as well as directions for future research. This review comprehensively summarizes information on L-theanine to provide an approach for an in-depth study of L-theanine production.