To obtain a 3D model of microscopic dynamic samples, a 3D microscopic imaging method based on a single objective and red-blue dual-apertures was proposed and a corresponding 3D microscopic specimen system for capturing images was established. In the system, the dual apertures covered with a red and a blue filters were respectively placed in the exit pupil plane of the objective. When it was illuminated by a white light, the color CCD in the imaging plane of objective could receive the perspective views of specimen expressed by red and blue images respectively at one exposure. The stereo image with an optical parallax for the specimen could be obtained by digital image processing, and the 3D model of the specimen could be further reconstructed by a phase matching algorithm. Experimental results indicate that the system has reconstructed the 3D model with a Central Depth Plane (CDP) up to 241.50 μm.As compared with double-objective 3D microscopic imaging systems, the proposed system is more compact and simpler and can obtain a 3D model of microscopic dynamic sample at one exposure, so that the system has important application prospects on microscopes and endoscopes.