The paper aims to study the effects of different fertilization treatments on photosynthetic characteristics, water use efficiency, yield and quality of mulberry leaves, and explore the optimal combined fertilizer application ratio suitable for the growth of protein mulberry. Three-year-old seedlings of ‘Yuyinsang-1’ were used as the materials. Eight treatments (T0, T1, T2, T3, T4, T5, T6 and T7) of nitrogen, phosphorus and potassium combined application were set to comprehensively screen out the optimal application rate for protein mulberry planted in Yulin wind-sand grassland area by measuring physiological characteristics, yield and quality of mulberry leaves. The results showed that different treatments of nitrogen, phosphorus and potassium combined application had significant effects on the photosynthetic characteristics of protein mulberry. With the increase of fertilizer application rate, chlorophyll content, net photosynthetic rate, transpiration rate and water use efficiency of mulberry leaves increased first and then decreased, and all physiological characteristics under T5 treatment were significantly higher than those of the control (P<0.05). Different treatments had no significant effects on plant height and stem weight per plant (P>0.05), but had significant effects on dry leaf weight per plant, biological yield and total yield per plant (P<0.05). The crude protein content increased first and then decreased, and the crude protein content of mulberry leaves under T4 and T5 treatments was significantly higher than that under the control treatment (P<0.05), and the crude fat content of mulberry leaves under T4, T5 and T6 treatments was significantly higher than that under the control treatment (P<0.05), which was 1.80, 1.90 and 1.78 times that of the control, respectively. The correlation analysis showed that the yield was very significantly and positively correlated with chlorophyll content, net photosynthetic rate, water use efficiency, crude protein content and crude fat content (P<0.01). The photosynthetic characteristics, water use efficiency, yield and quality indexes of protein mulberry were compared by membership function method, and T5 treatment (N fertilizer 150 kg/hm2, P fertilizer 90 kg/hm2 and K fertilizer 60 kg/hm2) had strong photosynthetic capacity, high yield, good quality, and high water use efficiency. Therefore, T5 treatment is the most suitable fertilizer application ratio in wind-sand grassland area in northern Shaanxi Province.