Actin filaments play essential roles in many kinds of cellular functions by interacting with hundreds of actin binding proteins. Here we probe the interaction between actin filament and a binding protein, alpha-actinin, using an atomic force microscopy (AFM) and dynamic force spectroscopy (DFS). The distribution of rupturing events including specific and non-specific interactions of actin filament/alpha-actinin and BSA/alpha-actinin were analyzed. The rupture force of the actin filament/alpha-actinin binding was significantly larger than that of the BSA/alpha-actinin non-specific interaction, and the peaks represent typical multiple parallel bonds. In addition, based on the rupture forces in different loading rate DFS experiments, the dissociation constant of actin filament/alpha-actinin binding was estimated. The value is in good agreement with a previously reported value obtained by optical tweezer measurement. We expect that the present method will be useful for interaction measurement of actin filaments and many kinds of binding protein.