This research presented an annealing strategy to identify hairpin DNA from coil DNA with the same base composition but different arrangements using electrospray mass spectrometry (ESI-MS).A series of single-stranded DNA were annealed with their complementary sequences,respectively.All the five pairs of hairpin DNA and coil DNA were unambiguously distinguished by ESIMS with annealing strategy.This research offers a potential method to probe the DNA structure by comparing with mass spectral characteristics.