The full-length cDNA sequence of Gly m Bd 28K was chemically synthesized and expressed in Escherichia coli (E. coli) BL21 (DE3) as an inclusion body under the induction of 0.2 mmol/L of isopropyl β-D-1-thiogalactopyranoside (IPTG). The purity of the recombinant protein was over 90% following Ni-nitrilotriacetic acid (Ni-NTA) affinity chromatography, and its molecular weight was 29.71 kDa. The polyclonal antibody (pAB) against Gly m Bd 28K was prepared and referred to as pAB-28K, and it exhibited high specificity for the protein in soybean meal. We established an indirect enzyme-linked immunosorbent assay (iELISA) using the pAB-28K and the recombinant Gly m Bd 28K protein to determine the Gly m Bd 28K content in soybean products. The R(2) value of the standard curve was 0.9910, the average relative standard deviation (RSD) was 16.93%, and the average recovery was 95.50%, which indicated that the iELISA was highly reproducible and accurate. Therefore, the pAB-28K and the iELISA provide valuable tools for the rapid and sensitive detection of Gly m Bd 28K in food and feed products from soybeans. This protocol meets the technical requirements for quality control and food safety as related to soybean.