(1) Background: This study aimed to evaluate the effects of organic fertilizer dose on soil nutrients, wolfberry fruit nutrient compositions, and fruit yields. (2) Methods: We conducted a two-year field trial in two typical fields with different fertility levels in the Qaidam area. Six treatments were applied to each field, including CK, M2 M4, M6, M8, and M10 (representing 0, 2, 4, 6, 8, and 10 kg organic fertilizer/plant, respectively) in the high-fertility field and CK, M3, M6, M9, M12, and M15 (representing 0, 3, 6, 9, 12, and 15 kg organic fertilizer/plant, respectively) in the low-fertility field. An ANOVA was used to determine the significant difference between treatments, and the LSD method was used for multiple comparisons of analysis of variance. (3) Results: In the high-fertility field, the application of organic fertilizer significantly affected the total nitrogen (N) content, mineral N storage, and soil organic matter content. The application of too much organic fertilizer significantly increased the soil’s EC value. In the low-fertility field, the effect of organic fertilizer application on soil nutrient enhancement differed significantly among soil layers but significantly increased the contents of total phenols, flavonoids, and amino acids in wolfberry fruit, and there was a significant trend of increasing wolfberry yield with increasing organic fertilizer application. (4) Conclusions: In the Qaidam area of the Tibetan Plateau, it is recommended to apply 2–4 kg commercial organic fertilizer/plant in the high-fertility wolfberry orchards while 9–12 kg in the low-fertility wolfberry orchards is recommended.