A small, simple intravital microcirculation multi-spectral imaging system was constructed. Orthogonal polarization spectral (OPS) imaging and liquid crystal tunable filter (LCTF) were used for intravital microcirculation multi-spectral imaging. LCTF has excellent imaging quality and low-power loss, without moving parts and image shift. It can provide continuously, rapid and random-access selection of any wavelength in the visible to near-infrared range. An ear of nude mouse was adopted as experiment specimen. The multi-spectral images of ear microvessel of the nude mouse were acquired by this imaging system in two ways (detection polarization angle 90° and 0°). Compared with detection polarization angle 0° way the images acquired by orthogonal polarization spectral imaging way(detection polarization angle 90°) had better contrast and more details. It is concluded that the experiment has proved that the orthogonal polarization multi-spectral technique can enhance the effect of observation of intravital microcirculation.