DNA molecules that encode a small, high-speed self-hydrolyzing deoxyribozyme are used as templates for rolling circle amplification (RCA) to produce single-stranded DNAs (ssDNAs) of single- and multiple-unit lengths. Including self-cleaving deoxyribozymes in RCA products can generate large amounts of ssDNAs with defined sequence and length as well as precise termini. We also demonstrate the use of this method to efficiently generate ssDNA size markers by using deoxyribozyme reaction conditions that permit partial processing.