The field experiment was carried out to study the effects of different growing stages and different gradient way of regulated deficit irrigation (RDI) on growth, photosynthetic characteristics, yield and quality in Isatis indigotica of the Hexi oasis. Field experiments were conducted from 2016 in the National Key Irrigation Experimental Station of Zhangye Water Resources Bureau in Central Hexi Corridor District, from numerous The Isatis indigotica seeds, bred by the Department of Chinese herbal medicine of Gansu Agricultural University, were used as tested materials. Two-factor randomized blocks were designed with nine treatments, including different growing periods and different gradient way of RDI. Each treatment had three plots as repeats in a completely random-sized design. In each growth period, the photosynthetic parameters of Isatis indigotica were determined, and after the harvest, yield and quality index were measured on fresh Radix Isatis. The results would provide a theoretical basis for reasonable planting and sustainable development of Isatis indigotica in Hexi oasis. The results showed that:(1) In the periods of vegetative stage and fleshy root growth, the water deficit significantly reduced the Isatis leaf net photosynthetic rate, transpiration rate and stomata conductance, and the declines increased with the raising water deficit regulation (WDR) level. (2) The highest yield was achieved under light WDR (7 342.05 kg/hm²) in the mature period of the regressed deficit, which was 5.32% lower than that of the full irrigation (P> 0.05). (3) The highest utilization efficiency of WDR (2.14 kg/m³), was followed by mild WDR in the flesh root maturity stage. (4) The quality of Isatis root was greatly improved especially in maturity period by mild WDR. The indigo, indirubin and (R,S)-goitrin content of Isatis root were improved by 0.09, 0.41 mg/kg and 0.007 8 mg/g,but content of polysaccharide reduced by 3.37 mg/g, respectively. Therefore, considering the yield, water use efficiency and quality, the fleshy root maturity could be implemented with RDI slightly, i.e., the relative water content of the soil was 60%~90% during this period, which could be used as the optimum way of cultivating irrigation.