Cell-based Surface Plasmon Resonance imaging (SPRi) with anti-endoglin VHH antibodies microarray on a gold-covered biochip was used to characterize the binding to endoglin expressed on the surface membrane of SC cells. SPRi showed the specific capture of SC cells by the immobilized anti-endoglin VHHs, but not of THP-1 cells that do not express endoglin on their plasmatic membrane. The SPRi reflectivity shifts with SC cells bound by anti-endoglin VHHs ranged from 3.59 to 5.54% (average 4.66 ± 1.18%) and with THP-1 cells from 2.23 to 3.05% (average 2.62 ± 1.32% (p < 0.001)) and the SC cell densities with anti-endoglin VHHs ranged from 417 to 747.7, (average 548 cells/2.28 × 105 μm2 ± 132). Anti-endoglin VHHs bind epitopes located in the extracellular domains of endoglin with different avidity as suggested by the differences in SC cell densities. The cell-based SPRi of anti-endoglin VHH microarrays can be used both in studies aimed at a better understanding of endoglin function in living cells and in VHH applications as nanoprobes.