Aerolysin could self assemble and oligomerize heptameric complex which insert into the lipid bilayer to form a nanopore.Due to the electrochemically confined effects,the aerolysin shows the high sensitivity in single molecule analysis of DNAs.Here,aerolysin was used to directly discriminate the single acetylene linked deoxyribonucleoside triphosphates from a single oligonucleotide without label or PCR operations.After modifying with acetylene derivative,a single oligonucleotide produced seven times longer blockade duration inside an aerolysin nanopore,increasing the blockage current amplitude by 7% and narrowing the current distribution by 44%.Therefore,the modification of the linear side chain of acetylene derivative could be further used to improve the temporal and spatial resolution in single DNA analysis via the aerolysin nanopore.