Stability is the major factor that enables the application of nanofluids in energy conversion systems. Excellent rheological and thermal conductivity is the key for nanofluids to be an ideal heat transfer medium. In this work, we investigated the stability of Fe3O4 nanofluids by four characterization techniques, mainly from the viewpoint of surfactant type and mass ratio, and systematically studied its rheological and thermal conductivity properties by different experimental methods and analyzed the behavior of magnetic fluid thermal anisotropy by mathematical methods. The experimental results show that the surfactant Tetramethylammonium hydroxide (TMAH) has the best stability effect and can keep the Fe3O4 nanofluids more than 120 h. And the best ratio is when the mass ratio of Fe3O4 to TMAH is 1:4. The surfactant Polyvinylpyrrolidone (PVP) has the greatest effect on nanofluids viscosity. The increase in TMAH mass leads to a Newtonian fluid behavior of the nanofluids at low temperatures and low shear rates, besides shifting its shear thinning towards the high temperature region. However, from the curve relationship between the shear rate and shear stress, the Fe3O4 nanofluids belongs to the expansion fluid among non-Newtonian fluids. The surfactant Sodium dodecyl sulfate (SDS) have the greatest effect on the thermal conductivity of Fe3O4 nanofluids. Theoretical calculations show that as the concentration increases, the rate of heat transfer in the direction of the parallel magnetic field is significantly greater than in the direction of the perpendicular magnetic field, while the rate of heat transfer in the absence of a magnetic field is somewhere in between.