Tea seedlings (Camellia sinensis) have well-developed root system with strong taproot and lateral roots. Compared with ordinary cuttings, it has stronger vitality and environmental adapt ability, thus facilitating the promotion of good varieties. However, there is less of detailed research on the rooting and germination process of tea seeds. In this study, matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to conduct non-targeted spatial mass spectrometry imaging of the main organs during growth of tea seedlings. A total of 1, 234 compounds were identified, which could be classified into 24 classes. Among them, theanine, as the most prominent nitrogen compound, was synthesized rapidly at the early stage of embryo germination, accounting for more than 90% of the total free amino acids in radicle, and then transferred to each meristem region through the mesocolonial sheath, indicating that theanine-based nitrogen flow plays a decisive role in the organ formation during the development of tea seedlings. Nutrients stored in the cotyledon were rapidly hydrolyzed to dextrin and 3-phosphoglyceraldehyde at the early stages of germination, and subsequently converted to other forms that provided carbon and energy for development, such as raffinose and d-galactose (glucose), which were mainly distributed in the growing zones of the root apex and the apical meristems of the stem. This study provides a new perspective on the synthesis and metabolism of substances during the development of tea seedlings and contributes to a better understanding of the biological characteristics of tea varieties.