The DNA base stack provides unique features for the efficient tong-range charge transfer. For the purpose of investigating the hole transfer process of individual DNA and the optical readout of DNA information at the single molecule level, we performed single-molecule detection of DNA hole transfer (DNA HT) using single-molecule fluorescence spectroscopy, We have established the single-molecule detection of DNA HT at the single molecule level based on the fluorescence generation by combining the oxidative reaction of the quencher molecule through DNA HT and the cancelation of FRET. This "signal-on" detection system makes it possible to detect DNA HT in individual DNA and the base-pair mismatch in the target DNA with high sensitivity and accuracy.