Accurate placement of pedicle screw (PS) is crucial in spinal surgery. Developing new real-time intra-operative monitoring and navigation methods is an important direction of clinical application research. In this paper, we studied the spectrum along the fixation trajectory of PS in frequency domain to tackle the accuracy problem. Fresh porcine vertebrae, bovine vertebrae and ovine vertebrae were measured with the near-infrared spectrum (NIR) device to obtain the reflected spectrum from the vertebrae. Along the fixation trajectory of PS, average energy from different groups was calculated and used for identifying different tissues and compared to achieve the optimal recognition factor. Compared with the time domain approach, the frequency domain method could divide the spectra measured at different tissue points into different groups more stably and accurately, which could serve as a new method to assist the PS insertion. The results gained from this study are significant to the development of hi-tech medical instruments with independent intellectual property rights.