Line-scanning hyperspectral imaging (LHSI) is known to have a higher acquisition rate but lower sectioning capability than point-scanning hyperspectral imaging. To further increase the axial imaging contrast of LHSI, structured illumination was integrated into line excitation to remove the off-focus and scattered on-focus fluorescence signals. In an unsectioned leaf, the imaging contrast can be enhanced by 8 times, while in sectioned mouse skin tissues, a 4.5-fold enhancement can be achieved. With a spectral resolution of 1.15 nm, the fluorophores with seriously-overlapped spectra was proved to be separated without cross-talk by applying linear unmixing to the recorded spectral information.