The invention belongs to the technical field of microimaging and provides a method, an optical module and a system for extended field depth three-dimensional nanoscale-resolution imaging. The method comprises the following steps of 1, building the optical module having a double-helix point spread function and defocusing grating multistage imaging properties, 2, carrying out imaging of a molecule needing to be detected by the optical module to obtain a double-helix image, 3, determining a horizontal position of the molecule needing to be detected according to positions of middle points of double-helix sidelobes of the double-helix image, and 4, determining an axial position of the molecule needing to be detected according to a rotation angle of a connection line of centers of the double-helix sidelobes of the double-helix image, and the positions of the middle points of the double-helix sidelobes. Through combination of double effects of the double-helix point spread function and the defocusing grating multistage imaging, the method extends a field depth and improves a resolution ratio. The method can be used for dynamic range imaging of subcellular fractions having any depth values in a whole cell, can produce dynamic function images of multiple moving molecules, and has an important meaning for high-level understanding of a rule and a relationship between a subcellular structure and a cell function change.