【Objective】 To establish a tissue culture system for Acer negundo ‘Aurea’ and provide a new large-scale vegetative propagation method, a screening method comprising the basic medium, optimal hormone combination of proliferation and rooting medium, and acclimatization and transplant was applied. 【Method】 Using hydroponic new shoots as explants, the effects of MS, WPM and B₅ mediums on the primary culture, 6-BA, TDZ and NAA on the proliferation culture, IBA and NAA on the rooting culture, and relative humidity on the transplant were investigated using a step-wise single factor selection. 【Result】 While both MS and WPM mediums were better than B₅ in inducing axillary bud sprouting, MS was superior to WPM for the shoot growth. The combined use of 6-BA, NAA and low-concentration TDZ effectively improved the proliferation coefficient of adventitious shoots. Each explant produced 5.8 new shoots in MS medium combined with 0.5 mg/L 6-BA, 0.01 mg/L TDZ and 0.06 mg/L NAA. Auxin significantly improved the rooting rate of proliferation shoots, and the rooting effect of IBA was better than NAA. A rooting rate of 100% was achieved in 1/2 MS medium with 0.05 mg/L IBA. The transplant survival rate was greater than 95% in a relative humidity of 85%. 【Conclusion】 The optimal basic medium was MS; the optimal proliferation medium was MS with 0.5 mg/L 6-BA, 0.01 mg/L TDZ and 0.06 mg/L NAA; and the optimal rooting medium was 1/2 MS with 0.05 mg/L IBA for new hydroponic shoots of A. negundo ‘Aurea’. Relative humidity was the key influence factor on the transplantation and not the transplanting substrate.