The relationship between the birefringence of Fe3O4 magnetic fluids and the ambient temperature (in the range of 20~80 ℃) at several different magnetic field strengths (lies in 0~200 mT) are investigated. The results show that similar dependence of birefringence on magnetic field strength and temperature happens for magnetic fluids with different concentrations. Moreover, the birefringence of the magnetic fluid is inversely proportional to the ambient temperature at fixed magnetic strength and proportional to the magnetic field strength at fixed ambient temperature. In a given temperature and magnetic field strength, the birefringence of the magnetic fluid with high concentration is larger than that with low concentration. The phenomena and results are analyzed carefully and the physical mechanism is clarified in detail.