DNA with repetitive sequence of green fluorescent protein (GFP) was prepared by rolling circle amplification with phi 29 DNA polymerase from a bacterial expression vector carrying a GFP gene. The prepared DNA was subjected to a cell-free coupled transcription-translation system and green fluorescence was observed, which shows that the template with repetitive sequence properly functioned in the reaction. The efficiency of the reaction conducted by the template with repetitive sequence was higher than those by linear template coding single gene or by circular plasmid.