The characteristic identification factor of the vertebra at different positions was studied by spectroscopic technology. The spectral measurement system consists of computer, the integrated hand drill with dual-fiber probe (each fiber′s core diameter is 200 μm and the distance between the center of two fibers is 0.5 mm), halogen light source (wavelength is from 360 nm to 2 000 nm) and fiber optic spectrometer (detection wavelength: 200~1 100 nm). The diffuse reflectance spectrum and the reduced scattering coefficient of biological tissue could be obtained at the same time. Pig′s vertebra was chosen as experimental subject to measure the diffuse reflectance spectrum and scattering coefficient of different bone tissue on pedicle screw placement pathway. The characteristic identification factor was defined by analyzing the peak values, the size of the area and the slope of the spectrum in specific wavelength. We find that different bone tissue′s spectrums show different characteristics. The change of the peak values was 1.77 and the change of the area values was 2.13 times higher than that of scattering coefficient, respectively. At the range of 495 nm to 505 nm, the slope of spectrum of cortical bone and cancellous bone was positive. At the range of 520~535 nm, the slope of spectrum of cortical bone was positive, and the slope of cancellous bone was negative. It can be concluded that the peak value, the area and the slope obtained by analysis of spectrum′s characteristics could distinguish the difference between cortical bone and cancellous bone.