High-throughput DNA sensors capable of detecting single-base mismatches are required for the routine screening of genetic mutations and disease. A new strategy for the electrochemical detection of single-base mismatches in DNA has been developed based on hole transport through DNA self-assembled monolayers. We prepared hole-transporting DNA self-assembled monolayers. Their photocurrent measurements were carried out using amperometory. Large photocurrent was observed when the DNA duplex which constituted a hole-transporting DNA self-assembled monolayer had a full-matched sequence, whereas the photocurrent for a DNA duplex containing a mismatched base pair was much smaller.