One major challenge of the life-science research is to study the subcellular structure and the interactions among organelles and molecules or different molecules in vivo. To develop a single molecule detection and tracking technology, which can real-time detect multiple biomolecules in living cells, is significant to the study on the molecular mechanism of the life process. We design and establish a microscopy imaging system based on the distorted grating and the double helix point spread function technologies, which can extend the depth of field and reach nano-positioning in three-dimensional space. The simulational results show that the system can achieve dynamic detection the 12 μm-depth sample with high localization accuracy. And the real-time positioning and tracking of dynamic biomolecules in living cells is achieved experimentally.