To quantitatively measure the number of magnetic beads and their binding biomolecules by a Giant Magnet Resistance(GMR) biosensor, the influences of the orientation of external magnetic field, the positions and the aggregation of magnetic beads on the biosensor signals should be considered. In this paper, the influences of three kinds of factors are simulated using Comsol software and the simulated results show that the signal amplitudes of the biosensor would be reduced by the tilt of external field, the deviation of bead position from the central area of sensor strips, and the aggregation of beads, especial the tilt of external field. When the tilt is 0.5°, the signal will decrease by 80%. Comparing with the simulated results, a GMR biosensor with the width of 5 μm is fabricated. The dependence of signal outputs on the coverage of magnetic beads is measured and results indicate that it is a linear relation with some deviations. Furthermore, the experimental result is 63 μV when the coverage of magnetic beads is 23.6%, which is smaller than the simulated result in 247 μV. Obtained results prove that both kinds of deviations are owing to the three factors. To measure the magnetic beads quantitatively, it suggests that the measuring conditions should be as fellows: making the external magnetic field perpendicular to the sensor plane; keeping the tilt angle unchanged during the measuring process; putting the magnetic beads to concentrate at the central area of the sensor strips; and avoiding the magnetic bead aggregation.