Energy migration between the identical chromophores is a necessary process in both natural and artificial photosynthesis. The distance and orientation dependence of energy migration have not been experimentally investigated in detail. Here we propose a method to investigate energy migration. Two fluorophores are introduced into one strand of a DNA duplex with a quencher placed opposite one of fluorophores. This design enables asymmetrization of identical fluorophores and allows one fluorophore to behave as an acceptor. The emission intensities and lifetimes decrease depending on the efficiency of energy migration. Distance and orientation dependence are successfully quantified, and the excitation energy migration efficiencies measured are in excellent agreement with those calculated based on Förster theory. We also demonstrate that multi-step energy migration among four fluorophores can be estimated from the theory. These results may provide a basis for design and preparation of efficient light-harvesting photonic devices and chemical probes.