Nitrogen (N) is the main factor to influence the growth and development of blueberry. To increase N utilization efficiency, it is critical to understand the mechanisms underlying their resistance to N deprivation and identify the genes and metabolites involved. In this study, two-year-old blueberry 'Sunshine' seedlings were treated with N deficiency (ND) and a control (normal N supply). Transcriptome and metabolome studies of blueberry leaves with varying N deficiency periods (30 days and 90 days) were performed. Transcriptome profiling result showed there was a maximum of 10677 differentially expressed genes (DEGs) in the comparison group of ND90 vs.CK0. The ND90 vs. ND30 comparison group had 5957 DEGs, demonstrating that the duration of ND treatment was a critical factor for gene alterations. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that there were 40, 92, 39, 31, 20, 33 DEGs in photosynthesis, starch and sucrose metabolism, glycolysis/ gluconeogenesis, the tricarboxylic acid cycle (TCA), N metabolism, and amino acid metabolism pathways, respectively. Metabonomics research revealed a total of 939 metabolites, primarily lipids, amino acids, carbohydrates and flavonoids. The combined analysis found that 151 DEGs and 13 differentially abundant metabolites (DAMs) were associated with the shikimate pathway (21 DEGs and 4 DAMs), the phenylpropanoid pathway (107 DEGs and 5 DAMs), and the flavonoid pathway (23 DEGs and 4 DAMs). These findings provide new information to improve the understanding of the molecular mechanisms driving blueberry adaptation to N-deficient settings and will help improve blueberry N use efficiency through breeding.