Abstract: Objective：To develop a multiplex flow method for the detection of antibodies against MICB (major histocompatibility complex class I chain related molecule B), and analyze the distribution frequency of MICB antibodies in healthy adults. Methods：Recombinant proteins of MICB were produced by insect cell expression system. The purified MICB molecules were coupled to Luminex beads. The linear range, specificity, precision and accuracy of the detection system were evaluated. A total of 500 samples of healthy individuals were tested by multiplex. Results：MICB antibody multiplex flow detection assay was successfully established with a linear range from 3.1 to 100.0 μg/mL. The linearly dependent coefficient was 0.995. The rate of recovery was from 97.4% to 99.6% in recovery tests. The coefficients of variation (CV) of intraassay were 3.4% and 2.9% in the concentrations of 30 μg/mL and 60 μg/mL, and the CV of inter-assay were 4.5% and 4.9% respectively. The assay was of high specificity without crossreaction with MICA. MICB-specific antibodies were found in 4 (0.8%) of 500 healthy adults. Conclusion：The multiplex flow assay for MICB antibody showed high sensitivity, stability and accuracy and may be useful for the further study on the effect of MICB antibodies in organ transplantation.