An intraoperative near-infrared-I and near-infrared-II multi-spectral fluorescent navigation system and method of using same includes a light source module for emitting white light and excitation light for illuminating tissue to be tested to generate an emission light. An optical information collection module includes a white light camera for collecting the white light image, and near infrared-I and near infrared-II fluorescence cameras for collecting the near infrared-I and near infrared-II fluorescence images. A central control module is coupled to the light source and the optical information collection modules. An image processing unit pre-processes the white light image, and the near infrared-I and near infrared-II fluorescence images, for de-noising and enhancement. The image processing unit performs a pseudo-color mapping on the images to obtain pseudo-color superimposed images of the near infrared-I and near infrared-II for a surgical region, and completes imaging of the intraoperative near-infrared-I and near-infrared-II multi-spectral fluorescent navigation system.