Abstract: Tetrastigma hemsleyanum Diels et Gilg has antitumor, antiviral, and fever-relieving properties. The main active components are flavonoids. It grows in shaded environments in forests, and its growth, development, physiological changes, and synthesis of active components are regulated by short-wavelength light of undergrowth. To explore the spatial and temporal responses of physiological mechanisms and flavonoid synthesis in T. hemsleyanum to short-wavelength light, blue or purple monochromatic light were selected as treatment light sources, and white light was used as the control for 7, 15, 30, 45, and 60 d. The total flavonoids content, flavonoid-related enzymes activities, and physiological and biochemical indexes, such as photosynthetic pigments, anti-resistance enzymes activities, malondialdehyde (MDA), and primary metabolic solubility, of leaves were measured to investigate the spatial and temporal responses of the flavonoid synthesis process and physiological and biochemical changes in T. hemsleyanum to short-wavelength light conditions. The results showed that: 1) short-wavelength light treatments regulated the changes in photosynthetic pigments contents; the control and blue light treatments increased photosynthetic pigments contents, but the purple light treatment significantly inhibited this effect. 2) Continuous monochromatic short-wave light treatments put T. hemsleyanum under mild stress, and the total flavonoids content was the highest with blue light treatment (137.75 mg∙g⁻¹ in leaves and 149.00 mg∙g⁻¹ in tubers). The phenylalnine ammonialyase activities in leaves and tubers (96.50 U∙g⁻¹ FW, 109.47 U∙g⁻¹ FW) and chalcone isomerase activity in leaves (65.17 U∙g⁻¹ FW) were highest under purple light treatment; while chalcone synthase activity in tubers was the highest with blue light treatment (46.76 U∙g⁻¹ FW). In addition, photosynthetic pigments, resilience, and soluble substances differed in their responsiveness to short-wave light, and both blue and purple light promoted the accumulation of osmoregulatory substances compared with white light. This study provided a theoretical basis and practical guidance for light regulation of T. hemsleyanum quality.