As a tree that is more than 100 years old, ancient trees are extremely precious natural resources. However, under the many adverse effects brought about by the rapid development of human society, growth has been severely restricted. Therefore, the protection and rehabilitation of ancient trees is an important task that should not be delayed and should be persisted for a long time. After long-term natural selection, there are indigenous microorganisms in their rhizosphere soil that are closely related to their growth and have unique functions. Inoculating the inter-root soil of ancient trees with dominant indigenous Arbuscular Mycorrhizal (AM) fungi could potentially be a new method for rejuvenating ancient trees. Two indigenous AM fungal agents (Claroideoglomus claroideum and Funneliformis mosseae) successfully screened and propagated in the rhizosphere soil of ancient Ginkgo biloba trees over 100 years old in urban Shanghai were used as test materials, and inoculated into ancient Ginkgo biloba seedlings by single and mixed application methods for verification experiments. The results indicate that the inoculation of indigenous AM fungi had a significant growth-promoting effect on the phenotype, aerial part, root morphology, plant nutrient accumulation and biomass of ancient Ginkgo biloba seeding. Meanwhile, the study also found that the inoculated treatments positively impacted various physiological indexes, including relative chlorophyll content, photosynthetic physiological condition, antioxidant enzyme activity, and metabolite accumulation in seedlings. As a result, the seedlings experienced overall positive growth. However, the growth process of the ancient Ginkgo biloba seedlings was affected to varying degrees by different indigenous AM fungi. Quantification and comprehensive ranking of the growth-promoting effects of different strains of agents on ancient Ginkgo biloba seedlings by the affiliative fuzzy function synthesis method. The results showed that mixed inoculation (Claroideoglomus claroideum+Funneliformis mosseae) was more effective in promoting the growth of ancient Ginkgo biloba seeding. This study initially demonstrated the potential and effectiveness of indigenous AM fungi in promoting and rejuvenating ancient trees. It has been initially proved that indigenous AM fungi have certain potential and effect in promoting the growth and rejuvenation of ancient trees, and the corresponding validation test will be carried out on the ancient Ginkgo biloba trees subsequently. The results of the study can promote the localised application of indigenous AM fungi in the field and provide new ideas and technical support for the conservation and restoration of ancient trees.