The dipole orientation of single-molecule plays an important role in improving the fluorescence collection efficiency and promises to have applications in super-resolution imaging, protein folding, and F rster resonance energy transfer between fluorophores. However, these applications are realized usually by precisely manipulating the orientation of the dipole moment of single molecules. Here, the dipole orientation of 1,1′-dioctadecyl-3,3,3′,3′,-tetramethylindodicarbocyanine (DiD) single molecules with the permanent dipole moment of 14.9 D is manipulated by using an external electric field of 3500 V/mm. Single DiD molecules are prepared by using mixed solvent of chloroform and dimethyl sulfoxide. The dipole orientation of single molecules is manipulated by an external electric field during the evaporation of solvent. The fluorescence of single molecules is measured by splitting the fluorescence collected by an objective into the S-polarized and P-polarized beams, and the fluorescence polarization of single molecules can be calculated by measuring the intensities of two orthogonal channels (I_S and I_P). The distribution of dipole orientation angle (α) for single DiD molecules in poly-(methyl methacrylate) (PMMA) film is analyzed statistically, and its changes are compared under different electric fields. It is found that the dipole orientation angle α of single DiD molecules in the PMMA film without applying electric field obeys a single-peak Gaussian distribution with the most probable value of 41°, which results from the fluorescence dichroism signal of the high numerical aperture objective. Applying a perpendicular electric field to the surface of single-molecule sample, the distribution of dipole orientation angle α of single DiD molecules can be still fitted by a single-peak Gaussian function with the most probable value of 44.2°. The dipole orientation of single DiD molecules under the perpendicular electric field changes little. However, by applying a parallel electric field to the surface of single-molecule sample, the dipole orientation angle α of single DiD molecules changes prominently. It obeys a two-peak Gaussian distribution with the most probable values of ～ 32° and 55.5°, indicating that the orientation polarization of the dipole moment occurs to the single DiD molecules in PMMA film. The dipole orientation of single polar molecules tends to the parallel electric field in this case.