A novel method of three-dimensional (3-D) information acquisition of biological micro-organism based on integral imaging and optical microscope is proposed.In this method,a microl ens array that matches with optical path is designed and a 3-D information acquisition system under weak il lumination is developed using imaging theory and the combination of geometrical optics.Compared with other me thods,the magnification is increased by several hundred times,meanwhile more subtle 3-D information of sam ple s is acquired,the eyepiece lens system is retention and the structure of microscope has not been broken,so the aberration can be eliminated more efficiently.Because of whole imaging for samples,the elemental images array can acquire the 3-D information of the sample,not only the surface information but also the internal structure informat ion from the scattered beam by use of the diffuse transmission element,and the information is plentiful.The elemen tal images arrays are acquired by use of high-sensitivity EMCCD.And then for the bi ological micro-organism experimental data of element images are acquired by this microscopic integral im aging device,the 3-D structure and information of the sample are analyzed qualitatively and it is efficient for this system to acquire the 3-D information of the biological micro-organism.