We report the construction of periodic DNA nanoribbons (DNRs) by a modified DNA origami method. Unlike the conventional DNA origami, the DNR scaffold is a long, single-stranded DNA of tandem repeats, originating from the rolling circular amplification (RCA). Consequently, the number of folding staple strands tremendously decreases from hundreds to a few, which makes the DNR production scalable and cost-effective, thus potentially removing the barrier for practical applications of DNA nanostructures. Moreover, the co-replicational synthesis of scaffold and staple strands by RCA-based enzymatic reactions allows the generation of DNRs in one pot, further reducing the cost. Due to their unique periodicity, rigidity, and high aspect ratio, DNRs are efficiently internalized into cells and escape from endosomal entrapment, making them potential nanocarriers for imaging agents and biological therapeutics. We demonstrated proof-of-concept applications of DNRs as an intracellular pH sensor and an efficient small interfering RNA delivery vehicle in human cancer cells.