The root of Ophiopogon japonicus has been used as a traditional Chinese medicine and also a functional food ingredient for a long time in China. In the present study, 17 different homoisoflavonoid compounds were identified in the root extract of O. japonicus by HPLC–DAD and LCMS/MS analyses. The antioxidant activity of the of chloroform/methanol (1:1, v/v), methanol and 70% ethanol extracts, and two major isolated homoisoflavonoid compounds (methylophiopogonanone A and methylophiopogonanone B) from O. japonicus root were investigated by various in vitro assays. Methylophiopogonanone B showed the highest antioxidant ability according to four antioxidant methods. Among the extracts, the chloroform/methanol extract which contained high amounts of homoisoflavonoids was found to exhibit the strongest antioxidant activity. The results showed that O. japonicus root can be regarded as a potential source of homoisoflavonoids and natural antioxidant.