We developed the ancestral design method that incorporates a multiple amino acid sequence alignment of homologous proteins and construction of the related phylogenetic tree to infer the ancestral sequence. Ancestral residues were introduced into several thermophilic proteins, producing mutant proteins with further enhanced thermostability. We also predicted, synthesized, and characterized the complete ancestral sequences of B subunit of DNA gyrase. The ancestral protein showed thermostability similar to a modern thermophilic protein. Thus, the ancestral design will serve as an effective method for creating thermally stable proteins that only relies on the amino acid sequences of homologous proteins.