A system for measuring polarized backscattering differential spectrum is reported. In the system, linearly polarized light is used to illuminate the sample surface, parallel and perpendicular components of its backscattered light are measured with polarizer and spectroscope, thereafter, the differential spectrum is calculated. Our system is validated by the comparison with Monte Carlo simulation, in which two kinds of polystyrene spheres with diameters of 5.0 μm and 9.0 μm are used. Based on this, the influence of the distribution of sphere size on differential spectrum is studied initially and roughly. Its result shows the differential spectrum can reflect the change of size distribution sensitively. In the mixtures of small and large spheres, when the relative concentrations of large spheres is increased in limited range, the oscillation frequency keeps identical to the small spheres, but differential signal intensity is weaken . It implies the polarized backscattering differential spectra can be used for pre-cancer detection potentially.