Various DNAs were employed as hosts to investigate the sequence-dependent formation of fluorescent Au nanoclusters (Au NCs) in aqueous solution. By comparison among hairpin DNAs (HP-DNAs) with a pristine stem segment and varied loop sequences, we found that the emission behavior of the HP-DNA-hosted Au NCs is dependent on the loop sequences. The most efficient host to produce fluorescent Au NCs is the cytosine loop. However, relative to the cytosine and guanine loops, the loop composed of thymine as well as adenine produces Au NCs with a much weaker emission. Additionally, the emission behavior of Au NCs hosted by the single-stranded DNAs (ss-DNAs) with an identical base composition to the corresponding HP-DNAs still exhibits a cytosine-rich dependence. The fully matched DNAs seem to be less efficient than the corresponding loop and ss-DNA structures. Furthermore, the emission properties of HP-DNA-hosted Au NCs can be modulated by the loop length. The sequence-dependent formation of fluorescent Au NCs is believed to be caused by differences in binding nucleophilicity of the DNA heterocyclic nitrogen and exocyclic keto groups to the hydrolyzed Au(III) species. This work demonstrates the role of sequence in producing Au NCs that could serve as promising fluorescent nanoprobes in biosensing and DNA-hosted Au nanomaterials.