AbstractArrays of individual molecules can combine the advantages of microarrays and single-molecule studies. They miniaturize assays to reduce sample and reagent consumption and increase throughput, and additionally uncover static and dynamic heterogeneity usually masked in molecular ensembles. However, realizing single-DNA arrays must tackle the challenge of capturing structurally highly dynamic strands onto defined substrate positions. Here, we create single-molecule arrays by electrostatically adhering single-stranded DNA of gene-like length onto positively charged carbon nanoislands. The nanosites are so small that only one molecule can bind per island. Undesired adsorption of DNA to the surrounding non-target areas is prevented via a surface-passivating film. Of further relevance, the DNA arrays are of tunable dimensions, and fabricated on optically transparent substrates that enable singe-molecule detection with fluorescence microscopy. The arrays are hence compatible with a wide range of bioanalytical, biophysical, and cell biological studies where individual DNA strands are either examined in isolation, or interact with other molecules or cells.