A long one-dimensional single-stranded DNA (ssDNA) molecule with a periodic sequence motif is an attractive building block for DNA nanotechnology because it allows the positioning of oligonucleotide-labeled particles or molecules with high spatial resolution via molecular self-assembly simply by hybridization reactions. In vitro enzymatic isothermal rolling circle amplification (RCA) produces such long concatemeric ssDNA molecules. These are complementary in sequence to their circular template. In this chapter, the preparation of stretched and surface-attached RCA products at the single molecule level is described. The methods presented comprise the enzymatic circularization of a ssDNA oligonucleotide, the covalent coupling of amino-modified primers to carboxylated fluorescence beads, the preparation of a hydrophobic glass substrate, the RCA in a flow-through system, the postsynthetic staining and stretching of the RCA products as well as the microscopic observation of individual ssDNA molecules.