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The fabrication of peptide microarrays using hyperbranched polymers (HBPs) to improve the signal-to-noise ratio was demonstrated. Due to a high density of reactive groups at the chain ends of the HBPs, as well as to their spherical shape, HBPs can be used as linkers to increase the amount of immobilized peptides through raising the specific surface area of the glass substrate. A zwitterionic HBP was used as a blocking agent to reduce the noise level of the peptide microarrays. The zwitterionic HBP shows comparably excellent blocking ability to a commercially available BSA-based blocking agent. Thus, it was concluded that HBPs have high potential for the fabrication of highly sensitive peptide microarrays.