In the past, magnetoresistive sensor based biosensors, using superparamagnetic particles, have shown to be promising candidates for highly sensitive biosensors. These sensors can detect a single micron-sized magnetic particle. For single particle detection, research groups have developed models to predict the signal per particle. In these models, the separation distance plays an important role for the quantitative determination of the signal. However, mostly only the passivation layer thickness is included as the separation distance. In this paper, we describe a detection system based on a magnetic spin-valve sensor that is capable of giving position-time information of the magnetic behavior of one single bead. The results obtained with this system for the detection of a single particle signature are then compared with simulations. For this comparison, we developed a model where an additional particle-substrate separation distance is included. This distance is determined by a force balance of the perpendicular forces acting on the magnetic particle, including the magnetic and electrostatic force. These simulations agree well with the single particle detection experiment. (c) 2006 American Institute of Physics.