Oblique-incidence reflectivity difference (OIRD) is a novel real-time, label-free, and nondestructive optical detection method and exhibits encouraging application in the detection of antibody/DNA microarrays. In this study, for the first time, an OIRD label-free immunoassay was achieved by using adherent live cells as the probe. The cells were cultured on glass cells, and the affinity binding of antibodies targeted on the HLA class I antigen of the cell surface was detected with an OIRD. The results show that an OIRD is able to detect the binding process of anti-human HLA-A, B, and C antibodies on MDA-MB-231 cells and HUVEC cells. Control experiments and complementary fluorescence analysis confirmed the high detection specificity and good quantitative virtue of the OIRD label-free immunoassay. Label-free OIRD imaging analysis of cell microarrays was further demonstrated successfully, and the underlying optical mechanism was revealed by combining the theoretical modeling. This work explores the use of live cells as probes for an OIRD immunoassay, thus expanding the potential applications of the OIRD in the field of pathological analysis, disease diagnosis, and drug screening, among others.