Blackberries are important economic fruits, and they not only have a distinctive flavor but are also rich in nutritional and medicinal value. With the aim of elucidating the effects of different NH4+:NO3− ratios on blackberry fruit quality, the physiological characteristics and metabolomics of blackberry fruits were investigated in this study. The results revealed that the NH4+:NO3−/50:50 ratio treatment showed the maximum values for single fruit weight, transverse diameter, longitudinal diameter and the solidity–acid ratio of blackberry fruit as well as the maximum flavonoid and ellagic acid contents. The NH4+:NO3−/75:25 ratio treatment exhibited the maximum fructose, sucrose, glucose and anthocyanin contents and the highest superoxide dismutase (SOD) and catalase (CAT) activities. Moreover, a total of 857 known metabolites were detected in the blackberry fruits. Interestingly, NH4+ could increase the levels of amino acids and metabolites belonging to the indole and derivative classes in blackberry fruits, and D-proline, cysteine, tryptophan and indole may play vital roles in the formation of blackberry fruit quality. To produce blackberry fruit that is larger and has better flavor, a 50:50 ratio of NH4+:NO3−is recommended, while when the goal is higher anthocyanin and amino acid content, a 75:25 ratio of NH4+:NO3− is more appropriate. In addition, a 100:0 ratio of NH4+:NO3− is best for fruit storge. This study serves as a reference and provides technical support for the application of N to improve fruit quality and antioxidant capacity during small berries cultivation.