One-year-old peach potted seedlings were used as test materials. The base fertilizer was urea 3 g (of which ¹⁵N was labeled 0.4 g) and potassium dihydrogen phosphate 3 g. The CK was set as control. The treatment T1, T2, and T3 were added with 10, 80 and 150 mg γ-polyglutamic acid (γ-PGA) to investigate the effects of different concentrations of γ-PGA on peach seedlings growth and nitrogen uptake and utilization. The results showed that the activity of soil urease and catalase as well as the content of soil alkali-hydrolyzed nitrogen could be significantly increased by applying medium and high levels of γ-PGA. Application of medium and high amount of γ-PGA could promote root growth of peach seedlings, especially the growth of fine roots. Compared with the control group, the total root length, the number of branches, the number of root tips, the number of crossings, and the total root surface area increased by 51.95%, 40.53%, 30.72%, 35.21% and 45.23%, respectively. The net photosynthetic rate, the chlorophyll SPAD value, and the dry matter accumulation of plants could be significantly increased by applying medium and high γ-PGA. The activity of root system, the nitrate reductase, the rice straw transaminase, and the rice third transaminase were significantly increased by applying medium and high γ-PGA. Compared with the control group, the nitrogen uptake efficiency and nitrogen residual rate were increased by 27.80% and 27.07%, the nitrogen residual rate was increased by 14.00% and 19.04%, and the nitrogen loss rate was decreased by 16.43% and 19.49%, respectively. It can be seen that applying γ-PGA can improve soil physical and chemical properties in peach root area, improve nitrogen uptake efficiency and soil nitrogen residual rate, reduce nitrogen loss rate, and promote the growth of peach seedlings.