The second harmonic generation (SHG), as a nonlinear optical effect, has a wide range of applications in obtaining information such as material composition, structure, and properties due to its good polarization sensitivity. Although SHG spectroscopy or SHG microscopy has been used to explore the precise positioning or tracking of nanowires, there are few reports on the combination of SHG spectroscopy and SHG microscopy to study the structure of nanomaterials and the spatial orientation of crystal axes. In this work, we investigate the spatial orientation and crystal axis orientation of cadmium sulfide (CdS) nanowires by combining SHG spectroscopy and microscopic imaging. Firstly, we experimentally and theoretically study the spectral intensity of the SHG of CdS nanowires with the polarization direction of the incident light based on the all-optical analysis method proposed by the predecessors. We also analyze the influence of the azimuth angle of the crystal axis γ, ωand φon the pattern of the SHG of CdS nanowires in detail. Secondly, through the mutual verification of theoretical calculations and experimental measurement results, we successfully determine the three axial orientations of a single CdS nanowire. Finally, we also investigate the spatial orientation of a single CdS nanowire by using the polarization-dependent SHG microscopic imaging method. It is shown that different parts of the CdS nanowire have different SHG responses when the polarization is changed. These results provide a new idea and an important reference for studying the application of SHG spectroscopy and microscopic imaging in the research of high-precision spatial positioning of nanomaterials. This study provides important enlightenment for realizing the potential applications of nanomaterials in biomedicine.