The present paper puts forward multi-wavelength pulse oxygen saturation measurement based on dynamic spectroscopy to do the non-invasive determination of oxygen saturation. Compared to conventional ways, the new method makes full use of more wavelengths light and improves the measurement accuracy. During the experiment, the in-vivo measurements were carried out on 60 patients and their spectroscopic data were collected by the high sensitivity type fiber optic spectrometer. Single-trial estimation method was used to extract the dynamic spectroscopy at the wavelengths of 606.44~987.55 nm. Oxygen saturation obtained from arterial blood gas analysis is regarded as the true value. Synergy interval partial least square (siPLS) was used to establish the calibration model of subjects’ oxygen saturation values against dynamic spectroscopy data. The relative error of prediction is ±0.017 6, but the relative error of the subjects in the same set measured by the patient monitor which was two-wavelength measure system is ±0.116 4. Measurement results show that the use of the high sensitivity type fiber optic spectrometer to collect multi-wavelength spectroscopic data and dynamic spectroscopy method to process data can do better in improving the accuracy of the oxygen saturation measurement.