To study the optimal amount of nitrogen application for blackberry plants, different amounts of ammonium sulfate were applied to three-year-old healthy ‘Shuofeng’ blackberry plants at three key growth stages (early bud burst, the flowering stage and the fruit veraison stage) to explore the effects of nitrogen fertilizer application level on the physiological characteristics of leaves, yield and fruit quality of blackberry. Overall, the leaves turned yellow, and cellular metabolism was disrupted in the CK group (no nitrogen fertilization). Among the nitrogen treatments, the leaf chlorophyll content, photosynthesis efficiency and antioxidant enzyme activity were significantly higher under the T2 (40 g plant−1) and T3 (60 g plant−1) treatments than under the T1 (20 g plant−1) and T4 (80 g plant−1) treatments, while the contents of ascorbic acid, glutathione, hydrogen peroxide and malondialdehyde showed the opposite trends. The yield, single-fruit weight, solids–acids ratio and anthocyanin contents of blackberry fruits increased first and then decreased with increasing nitrogen application. The contents of polyphenols and ellagic acid in fruits decreased with increasing nitrogen application. Correlation and principal component analysis showed that yield and its components were closely related to leaf photosynthesis characteristics and the antioxidant system (P<0.05). Application of 20 g ammonium sulfate per plant at early bud burst, the flowering stage and the fruit veraison stage was profitable, which resulted in more vigorously growth, increased yield and fruit quality, while the production cost was the most economical because of the low fertilizer consumption.