Measuring the persistence length of single-stranded DNA (ssDNA) is a challenge that has been studied by different methods lately. The short persistence length and the appearance of stem loops complicate the measurement and leads to large variability in the measured values. Here we describe a DNA origami-based method for studying the persistence length of ssDNA. By folding a DNA origami structure that consists of two rigid rods with an ssDNA segment between them, we developed a method to measure and characterize the persistence length of ssDNA. We image the structure with an atomic force microscope (AFM), while the distance between the rods' ends provides the end-to-end distance of each ssDNA segment. By fitting the measured distribution to the ideal chain polymer model, we measured a persistence length of . This method enables one to measure short or long strands of ssDNA, the influence of repetitive nucleotide sequences, the effect of environmental conditions and the interaction of proteins with ssDNA.