Abstract: To study the effect of fermented manure on the contents of physiological active substances in Ginkgo biloba L., under the field condition, 3 years old Ginkgo biloba L. seedlings were used as test materials, 5 treatments including control (T1), conventional fertilization (T2), potassium fertilizer (T3), nitrogen fertilizer (T4), fermented manure (T5) were set up, 3 repeats were conducted. The results showed that: The chlorophyll a content of T5 treatment increased by 80.36%, 65.89%, 62.32%, 71.53%, 72.59%, respectively, compared with the control; the chlorophyll b content of T5 treatment from May to August increased by 16.67%, 14.06%, 18.18%, 18.52%, compared with T2; anthocyanin content of T5 treatment increased by 5.19%, 9.54%, 6.99%, 6.92%, 8.79%, compared with T2; flavonoid content of T5 treatment increased by 21.05%, 20.49%, 23.46%, 28.57%, 23.61%, compared with T2; soluble sugar content of T5 treatment increased by 22.39%, 25.58%, 20.43%, 20.00%, 23.44%, compared with T2. T5 was significantly higher than that of the control; soluble protein content of T5 treatment increased by 15.90%, 18.26%, 21.71%, 21.66%, 26.02%, compared with T2. In conclusion, T5 is significantly better than the control and conventional fertilization in improving the contents of physiological active substances in Ginkgo biloba L..