The 3D networks of cytoskeletons are mechanistic bases for cell and tissue function (e.g. epidermal barrier function). To examine the details of the 3D networks, we extended multi-target super-resolution microscopy IRIS (Kiuchi et al., Nat. Methods 12: 743-746, 2015) to 3D imaging. IRIS uses exchangeable probes that directly associate with and dissociate from their targets. By integrating single-molecule localization and sequential labeling, IRIS enables the high resolution imaging of multiple targets within a single specimen. By the repeated associations of the probes, the loss of fluorescent signals due to photobleaching during the 3D imaging can be compensated. Moreover the labeling density on the target structures can be unlimitedly increased, yielding greatly improved continuous labeling along the cytoskeletal filaments. We developed IRIS probes for three cytoskeletons and focal adhesions from the target-associated proteins. For 3D IRIS imaging, we combined the IRIS method with HILO illumination and astigmatism using adaptive optics. This allowed us to discern microtubules at heights of 6.3 μm and 6.5 μm in the apical region of the cell. Thus IRIS is a potent tool to monitor the 3D network of cytoskeletons.