This paper investigates dynamic characteristics of a historic wooden structure by ambient vibration testing, presenting a novel estimation methodology of story stiffness for the purpose of vibration-based structural health monitoring. As for the ambient vibration testing, measured structural responses are analyzed by two output-only system identification methods (i.e., frequency domain decomposition and stochastic subspace identification) to estimate modal parameters. The proposed methodology of story stiffness is estimation based on an eigenvalue problem derived from a vibratory rigid body model. Using the identified natural frequencies, the eigenvalue problem is efficiently solved and uniquely yields story stiffness. It is noteworthy that application of the proposed methodology is not necessarily confined to the wooden structure exampled in the paper.