The production costs for monoclonal antibodies (MAbs) utilized in medical diagnostic kits are inevitably high because the MAbs are mostly obtained from hybridoma cell culture. Here, we report the development and validation of a novel affinity silk protein produced by transgenic silkworm technology as a possible alternative diagnostic tool for cancers. We generated a transgenic silkworm expressing a cDNA construct containing fibroin L-chain fused to a single-chain variable fragment (scFv) derived from a MAb against carcinoembryonic antigen (CEA). The transgenic cocoons were dissolved in aqueous lithium bromide solution, applied to 96-well plates, and analysed by enzyme-linked immunosorbent assay. The scFv-conjugated affinity silk protein specifically recognized CEA as well as the parental MAb. The binding activity was retained after several months of storage in coated plates or concentrated solution. Thus, the scFv-conjugated affinity silk protein provides a potentially useful alternative to conventional MAbs in medical diagnostic kits.