Study of long single-stranded telomeric DNA is important for a variety of basic science and biotechnological applications, yet few methods exist for synthesis and visualization of single copies of this DNA in solution at biologically relevant length scales necessary for assessment of heterogeneity in its structure and behavior. We have synthesized kilobase-long single-stranded human telomere mimics in situ by rolling circle replication (RCR) on a microscope coverslip surface and visualized individual strands by staining with SYBR Gold. Under buffer flow, differential extensibility and varying morphology of these long telomere-mimicking DNA sequences were observed at the single-molecule level in real time. Using this procedure, we detected striking differences in the extensibility of individual RCR products based on the human G-rich telomeric sequence in the presence and absence of short, complementary single-stranded oligonucleotides. We also apply this new mode of single-stranded DNA characterization to probe the interaction of kilobase-length telomere mimics with the small-molecule G-quadruplex-binding agent TMPyP4.