Mirror-image proteins (d-proteins) are promising scaffolds for drug discovery because of their high proteolytic stability and low immunogenic properties. Facile and reproducible processes for the preparation of functional d-proteins are required for their application in therapeutic biologics. In this study, the author designed and synthesized novel monobody variants with two cysteine substitutions that facilitate the synthetic process via sequential native chemical ligations. The synthetic anti-GFP monobody in initial model study exhibited good binding affinity to the target enhanced green fluorescent protein. In vivo administration of the mirror-image anti-GFP monobody to mice induced no anti-drug antibody production. The structure–activity relationship study of anti-d-MCP-1 monobody revealed that cysteine substitutions at two appropriate positions in combination with cysteine-selective chemical stapling improved the thermal stability of the scaffold while keeping the high-affinity target binding. These results highlight that the synthetic d-monobody variants are functional protein scaffolds with low immunogenic properties.