A novel molecular hyperspectral imaging (MHSI) system based on AOTF (acousto-optic tunable filters) was presented. The system consists of microscope, AOTF-based spectrometer, matrix CCD, image collection card and computer. The spectral range of the MHSI is from 550 to 1 000 nm. The spectral resolution is less than 2 nm, and the spatial resolution is about 0.3 μm. This paper has also presented that spectral curves extracted from the corrected hyperspectral data of the sample, which have been preprocessed by the gray correction coefficient, can more truly represent biochemical characteristic of the sample. The system can supply not only single band images in the visible range, but also spectrum curve of random pixel of sample image. This system can be widely used in various fields of biomedicine, clinical medicine, material science and microelectronics.