Birefringent effect dependent on magnetic intensity and wavelength in a water-based Fe3O4 magnetic fluid over the wavelength range of 400–700 nm is studied in this letter. The results are compared with the theory proposed by Llewellyn [J. Phys. D 16, 95 (1983)], in which the optical anistropic properties of the magnetic fluid due to the aggregation of the particles at a relatively low density is suggested. It is shown that the degree of aggregation corresponds to the value of the axial ratio of the aggregated particles. So does the magnitude of the birefringence.