A noncollinear acousto-optic tunable filter (AOTF) was designed with a comprehensive treatment of the properties of TeO 2 as an acoustooptic (A-O) material. The results of optical testing validated that it predicted the performance of the designed AOTF. The bandwidth of the AOTF was very narrow in the visible light range. The high spectral resolution of AOTF was useful in practical applications to the hyperspectral imaging. The experimentally observed spectral pattern of the diffracted light was nearly the same as the theoretical result. The measured tuning relationship between the diffracted central optical wavelength and acoustic frequency was in accordance with the theoretical prediction. It demonstrates the accuracy of our design theory. Furthermore, by selecting the AOTF as the spectroscopic element, a hyperspectral microscopic imaging system was designed. The hyperspectral microscopic images of the rat skin tissue under the different optical center wavelength were acquired. The experimental results indicated that the imaging performance was satisfactory. The stratums of the rat skin can be clearly distinguished. The inner details of the epidermis and the corium can be shown on the hyperspectral microscopic images. Some differences also can be found by the comparison of the hyperspectal images under the different optical wavelengths. The study indicated the applicability and the advantage of our system on biomedicine area.