The objective of this study was to develop a purified soy isoflavone product which is potentially efficacious for relieving estrogen-deficient diseases (i.e. menopausal symptoms). In this study, a simple method was developed for producing isoflavones from soybean flour including only three steps extraction, hydrolysis and crystallization. Then the extraction and purification parameters were optimized to give a high yield of total isoflavones, about 0.66 mg aglycones/g soybean flour, which is more than two times of the initial yield. HPLC analysis and MTT cell proliferation assay using MCF-7 cells revealed that the product thus obtained not only contained a high content of isoflavone aglycones but also had estrogenic activity. MTT data also revealed that both genistein and daidzein exhibited estrogenic effects at lower concentrations and anti-proliferative effects at higher concentrations, and 1 µM genistein and 10 µM daidzein exerted significant estrogenic activities which were not more than that of endogenous level of 17beta-estradiol (E2). The production method developed can be used as guideline for manufacturing soy isoflavones, and MTT assay was demonstrated to be suitable for quality control on isoflavone products, and the results on the estrogenic properties of isoflavones can be used as reference data for their effective and safe usages in estrogenic therapy.