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• Single-layer three-dimensional structured illumination microscope (SL3D-SIM) can achieve fast continuous imaging of living cells at certain depth. • The reconstruction algorithms for SL3D-SIM were mostly constructed on the Wiener-SIM framework which is complex and time-consuming. • Joint space-frequency reconstruction (JSFR) framework establishes a fast reconstruction method for SL3D-SIM. • A 90 times improvement of the reconstruction speed to that of the conventional method. Although three-dimensional structured illumination microscopy (3D-SIM) that enhances the spatial resolution of widefield microscope in three dimensions has been successfully commercialized as a variety of products, the low imaging speed impedes its application in fast subcellular dynamics. To adapt to fast dynamics, most 3D-SIM setups provide an option for the users by canceling the axial scanning during the acquisition, by which two-dimensional optically-sectioned, super-resolved (OS-SR) time series can be obtained with single-layer 3D-SIM data. However, the conventional reconstruction workflow implemented in the frequency domain generally takes seconds for every single reconstruction, which nullifies real-time feedback for these 3D-SIM setups. In this paper, we establish a fast single-layer reconstruction approach for 3D-SIM by extending a high-speed reconstruction framework, joint space-frequency reconstruction (JSFR), to single-layer 3D-SIM. Consequently, the reconstruction speed is improved over the conventional method, without compromising the image quality. This approach is expected to provide a useful tool for 3D-SIM microscope manufacturers and their users to achieve real-time, single-layer imaging of living cells. [ABSTRACT FROM AUTHOR]