Tetrastigma hemsleyanum is a valuable medicinal plant whose dryland cultivation typically yields 30-35% lower flavonoid concentration than forest understory systems due to soil and microbial deficiencies. We investigated whether biochar amendment could bridge this quality gap through rhizosphere microecological regulation. Using a split-plot pot experiment with in situ soils from a bamboo forest and a vegetable field, we applied biochar at 2%. Biochar in bamboo forest (MBBC) achieved the highest flavonoid concentrations, exceeding unamended forest and vegetable controls by 22% and 35%, respectively. Biochar effects were habitat-specific. In acidic forest soils (pH 4.95), it raised the pH to 5.61, while in vegetable fields, it boosted leucine aminopeptidase by 159%. Partial least squares path modeling revealed biochar exerted its effects indirectly (indirect effect = 0.88), with soil extracellular enzymes mediating between soil conditions and plant biosynthetic enzymes (PAL, CHS, CHI). Fungal composition was positively associated with biosynthesis (beta = 1.68, p < 0.01), particularly Mortierellomycetes, whereas bacterial diversity unexpectedly exhibited a significant negative correlation with it (beta = -0.79, p < 0.05). Biochar disrupted Eurotiomycetes dominance in forest soils (from 85% to 39%), creating functionally diverse niches that were associated with enhanced flavonoid accumulation. These findings demonstrate biochar functions as an ecological niche regulator, providing a sustainable strategy for high-quality medicinal plant production in non-native habitats.