The seed of tree peony and herbaceous peony contained a variety of stilbenes which possess many pharmacological activities, such as antitumor, anti-inflammatory, allergy and neuraminidase inhibition. In order to develop and use peony seed resources, a simple and sensitive HPLC-DAD was developed for simultaneous determination of 10 stilbenes in peony samples, i.e.,suffruticosol A,suffruticosol B,suffruticosol C,trans-resveratrol,cis-ε-viniferin,trans-ε-viniferin,cis-suffruticosol D,cis-gnetin H,trans-suffruticosol D and trans-gnetin H. At the same time, the scavenging activity of DPPH free radicals was employed to evaluate their antioxidant effect. The results showed that the 10 stilbenes are mainly present in peony seed coat (total content of more than 16.7%) compared to peony seed kernel (total content less than 0.3%), and can be enriched in the extract of peony seed coat (total content of more than 75%) The extract of peony seed coat and 10 stilbenes exhibited significant antioxidant properties. This work provides a foundation for comprehensive utilization of the tree peony and herbaceous peony seed resources.