Nitrogen (N) is the most critical element influencing plant growth and development. Different plant species exhibit varying preferences for different N forms. In order to identify an appropriate nutrient solution N formula for optimizing blueberry substrate cultivation, we investigated the effects of seven different NH₄⁺-N/NO₃⁻-N ratios on the growth characteristics, photosynthetic physiology, mineral element content, enzymes related to N metabolism, and fruit quality, with ‘F32’ used as the experimental material and water served as controls. The results demonstrated that both the aboveground and belowground parts of blueberry plants exhibited enhanced growth when NH₄⁺-N was used as the primary N source in the nutrient solution, compared to single NH₄⁺-N or a high NO₃⁻-N ratio. The most significant growth promotion occurred when the NH₄⁺-N to NO₃⁻-N ratio was 7:3. When NH₄⁺-N and NO₃⁻-N are concurrently supplied in the nutrient solution, the processes of NO₃⁻ reduction, the GS-GOGAT cycle, and NH₄⁺ assimilation are significantly enhanced during nitrogen metabolism. Thereby, providing a theoretical foundation for optimizing nutrient solution management in substrate-cultivated blueberry.