Procyanidin from grape seed were separated and purified by the aqueous phase two-extraction. Aqueous two-phase extraction systems, differently assisted extraction methods and the extraction strategies were firstly studied. The best extractions of procyanidin by aqueous two-phase system were investigated based on orthogonal experimental design. The results showed that, in the aqueous two-phase extraction system of ethanol and (NH₄)₂SO₄, the extraction method was the microwave-assisted extraction, i.e., Ethanol, water, and compounded by microwave treatment, as well as (NH₄)₂SO₄ were added. It was found that the best extraction conditions were 30%(w/w) ethanol/20%(w/w) (NH₄)₂SO₄, 3%(w/w) grape seed powders, pH=4.0, microwave power 100W, ,and microwave time 5 min. Under these best extraction conditions, the recovery rate of procyanidin was 94.1%, and its yield was 25.8 mg/g.