The present invention relates to a method for preparation of soybean leaves having a high content of an isoflavone derivative in a dark condition and soybean leaves having a high content of an isoflavone derivative prepared thereby. Specifically, treatment of a soybean plant 20 days to 60 days after seeding with a predetermined concentration of ethylene in a dark condition was found to accumulate higher concentrations of isoflavone derivatives in soybean leaves than treatment with ethephon in a light condition, which requires a high level of energy. Therefore, when used, the method of the present invention can economically and quickly prepare soybean leaves having a very high content of isoflavones, and the soybean leaves having a high content of isoflavone derivatives, an extract of the soybean leaves, and a fraction of the extract can be advantageously used as a food and medicine material against diseases caused by estrogen unbalance and deficient antioxidant activity.