A series of Fe3O4 particle chains with an average particle diameter of 150 nm and different lengths were synthesized by using the self-assembly method at reduced temperature in different synthesizing magnetic fields. The influence of synthesizing magnetic field on the properties of the magnetite particle chains was studied by structural analyses, magnetometry measurement, and ferromagnetic resonance. A uniaxial magnetic anisotropy in the saturation field (Hs), hysteresis loop, and ferromagnetic resonance were observed to increase with increasing synthesizing field. The saturation magnetization and g-factor were found to increase slightly with increasing synthesizing field. The demagnetizing fields and demagnetizing factors were determined from the experimental data of magnetometry measurement, ferromagnetic resonance, and also numerical calculation, which agreed reasonably well. It was found that the magnetization non-uniformity in the chains and the magnetostatic interaction among the chains have an important effect on the shape anisotropy of the chain assembly.