Persistence length measurement of single stranded DNA is viable by various tools. Most of them have been done by incorporation of the worm-like chain model. For example, two ends of single stranded DNA were tagged by donor and acceptor dyes to measure average end to end distance which is represented as FRET efficiency. It was converted to persistence length by comparing the FRET efficiency with calculated FRET efficiency based on models such as the worm-like chain model. The recent attempts to obtain persistence length is combination of fluorescence correlation spectroscopy (FCS) with HYDRO program. HYDRO program is simply noted as the upgrade of Stokes–Einstein equation. The Stokes–Einstein equation calculates diffusion coefficient (which is inversely proportional to diffusion time) by assuming the molecules as pure sphere. However, the HYDRO program has no limitation regarding to the shape of molecule. For estimation of single stranded DNA persistence length, the diffusion time of number of worm-like chain polymer was generated and its diffusion time is calculated by the HYDRO program which is compared with the experiment diffusion time of FCS. The polymer property was adjusted to find the optimal persistence length.