Abstract <p>In order to screen the appropriate nitrogen (N) fertilizer concentration during the seedling stage of blueberry cultivated with substrate, this study treated one-year-old ‘Star’ blueberry seedlings with 100%&#xa0;Hoagland nutrient solution containing 0 mM (CK), 2 mM (T1), 4 mM (T2), 8 mM (T3), 15 mM (T4) N concentrations, the effects on the growth and physiological indexes as well as the expression of related genes of blueberry seedlings were explored, and the correlation and principal component analysis were conducted on different indexes. The results showed that the application of N fertilizer significantly increased the dry and fresh weight of blueberry as well as the chlorophyll content, and notably promoted the growth of blueberry seedlings. The plants treated with T2 grew better with the maximum dry and fresh weight, plant height, average crown width, and chlorophyll content of leaves. Additionally, T2 significantly enhanced the expression of chlorophyll synthesis-related genes <i>VcCHLH1</i>, <i>VcCHLH2</i>, <i>VcCHLD1</i> and <i>VcCHLI1</i> and N&#xa0;utilization-related genes <i>VcNRT2</i> and <i>VcGS3</i> in blueberry seedling leaves. Correlation and principal component analysis revealed that plant height and average crown width were closely related to chlorophyll and soluble protein (SP) content, and superoxide dismutase (SOD) activity, while glutathione (GSH) and ascorbic acid (AsA) content played key roles in inhibiting the excessive accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). In summary, 4 mM N concentration (T2) is appropriate for seedlings of soilless cultivated blueberry. This study can serve as a theoretical foundation for the scientific management of nutrient solution in blueberry substrate cultivation.</p>