DNA hairpins are widely used to synthesize silver nanoclusters (AgNCs) with excellent optical properties due to their specific secondary structure. Hairpin-AgNCs have been popularly employed for sensoring applications, while no systematic study has been done about the effect of stem sequence on the fluorescence property of hairpin-AgNCs. In this presented work, the synthesizing conditions of hairpin-AgNCs were fully examined first. Then, the effect of percentage content and distribution of GC base pairs as well as stem length on the fluorescence property of hairpin-AgNCs were studied. Intriguing phenomena were observed and basic conclusions were drawn, which would be helpful to understand the hairpin-AgNCs comprehensively and instructional for the applications using hairpin-AgNC probes.