Isohemagglutinin assays employing red blood cells (RBCs) are the most common assays used to measure antibody titer in ABO-incompatible kidney transplantation (ABOi KTx). However, ABO antigens expressed on RBCs are not identical to those of kidney and antibody titers do not always correlate with clinical outcome. We previously reported that CD31 was the main protein linked to ABO antigens on kidney endothelial cells (KECs), which was different from those on RBCs. We developed a new method to measure antibody titer using a microarray of recombinant CD31 (rCD31) linked to ABO antigens (CD31-ABO microarray). Mass spectrometry analysis suggested that rCD31 and native CD31 purified from human kidney had similar ABO glycan. To confirm clinical use of CD31-ABO microarray, a total of 252 plasma samples including volunteers, hemodialysis patients, and transplant recipients were examined. In transplant recipients, any initial IgG or IgM antibody intensity >30,000 against the donor blood type in the CD31-ABO microarray showed higher sensitivity, specificity, positive predictive value, and negative predictive value of AABMR, compared to isohemagglutinin assays. Use of a CD31-ABO microarray to determine antibody titer specifically against ABO antigens expressed on KECs will contribute to precisely predicting AABMR or preventing over immunosuppression following ABOi KTx.