We present an optical biosensor technology that integrates the tethered particle motion technique and the magnetic tweezer technique. The goal is to quantify the three-dimensional mobility of bound particle labels and to characterize the bond between the particle and the surface. We show, using a series of four different lengths of dsDNA (105–590 bp), that plots of the height as a function of the in-plane particle position reflect the bond length and bond flexibility. We analyse ensembles of bound particles and show that the height displacement is at maximum the bond length, but that non-specific sticking causes large variations between particles. We also measured the height of bound particles under the influence of magnetic forces. A magnetic gradient force towards the surface brought particles on average closer to the surface, but a magnetic gradient force away from the surface did not bring all particles away from the surface. We show that the latter can be explained by magnetic anisotropy in the particles. Our results demonstrate that mobility detection of bound particle labels in an evanescent field is a promising technique to characterize the bond between a particle and a surface in a biosensor system.