We have previously reported that a protein library can be constructed by directly combining PCR amplification of a single DNA molecule and cell-free protein synthesis. To specifically amplify single DNA molecules, however, two-step PCR with nested primers was used. Here we describe a simpler method for single-step amplification of a single molecule. The method involves the use of both hot-startable DNA polymerase and a DNA template that has homo-priming sequences at both ends for amplification using a single primer. These two modifications greatly decreased the possibility of formation and subsequent accumulation, respectively, of primer-dimers that inhibit the amplification of target template. In addition, a high-fidelity DNA polymerase was successfully used, resulting in the significant reduction of the accumulation of mutations during amplification.