Nitrogen (N) is associated with amino acid metabolism in higher plants. Theanine is an important amino acid in tea plants. To explore the relationship between theanine metabolism and N conditions, we examined the differentially expressed genes (DEGs), proteins (DEPs), and microRNAs (DEMs) involved in theanine metabolism in tea plant shoots and roots under N sufficiency and deficiency conditions. Transcriptome, proteome, and microRNA analyses were performed on tea plant shoots and roots under N sufficiency and deficiency conditions. The contents of theanine, expression levels of genes involved in theanine metabolism, contents of proteinogenic amino acids, and activity of enzymes were analyzed. The DEP-DEG correlation pairs and negative DEM-DEG interactions related to theanine metabolism were identified based on correlation analyses. The expression profiles of DEGs and negative DEM-DEG pairs related to theanine biosynthesis were consistent with the sequencing results. Our results suggest that the molecular and physiological mechanism of theanine accumulation is significantly affected by N sufficiency and deficiency conditions. The DEGs, DEPs, and DEMs and the activity of the enzymes involved in theanine biosynthesis might play vital roles in theanine accumulation under N sufficiency and deficiency conditions in the shoots and roots of tea plants.