According to the existing Agrobacterium rhizogenes infection method, we used the leaves of Ginkgo biloba L. sterile seedlings as explants to optimize the experimental operation of Agrobacteriuminduced ginkgo hairy roots, and observe the growth of ginkgo hairy roots. The polyphenol content, relative expression of enzyme gene which related to polyphenol synthesis and POD gene in hairy root tissues on different days of subculture were determined to explore the physiological mechanism of browning of ginkgo hairy roots. The results show: (1) the optimal preincubation time of Agrobacterium rhizogenes infection was 3 d, the strain was R1601, the coculture medium was White, and the infection time was 30 min. The induction rate of Ginkgo biloba hairy root after optimization of infection operation reached 72.36%; the optimal IBA concentration for ginkgo hairy root growth was 0.5 mg/L. (2) The hairy roots of ginkgo have a milky white color when initial rooting and they are mostly clustered at the wound. These hairy roots are nondirectional when growing and can be induced at the wound both on the front and back of the leaves. The root length can reach 0.4-0.8 cm at 20 days after sterilization. After 25 days of subculture, the hairy roots are white. However, at 35 days of subculture, part of the hairy roots which from bottom to middle turned yellow. At the 45 days of subculture, most of the hairy roots turned from white to yellow, and browning began to appeared from the root to the middle. (3) The relative expression levels of PAL, C4H, 4CL and POD of hairy roots in 35 d after subculture were respectively account for 68%, 44%, 51% and 52% of 25 days, and the relative expression of PAL, C4H, 4CL and POD gene at 45 d after subculture were respectively account for 19%, 75%, 86%, and 105% of 35 days. (4) The total phenolic contents of ginkgo hairy roots at 35 and 45 d after subculture were 17.14 and 13.78 mg/g, respectively. The change of PAL gene expression was inconsistent with the expression of other two key enzyme genes (C4H, 4CL) at downstream, and the significant decrease of PAL gene expression did not affect the expression of C4H and 4CL gene, which indicate the change of PAL gene expression has little effect on the downstream synthesis rate, and it may not be the ratelimiting enzyme of the phenylalanine metabolic pathway of ginkgo hairy root; while the trend of C4H and 4CL gene expression is highly consistent, indicating that C4H should be one of the ratelimiting enzymes in this pathway; it is speculated that PPO may be involved in the early stage of browning reaction of hairy roots, which leads to the inhibition of hairy root growth and the increase of POD gene expression level, then bring POD participates in browning reaction.