Objectives <p>This study aimed to apply CUBIC tissue clearing combined with three-dimensional (3D) imaging to visualize the pathological architecture of systemic light chain (AL) amyloidosis in the human tongue, assess spatial and microstructural changes associated with amyloid deposition, and explore its potential to enhance early diagnostic accuracy.</p> Materials and methods <p>Seven tongue specimens from confirmed AL amyloidosis cases underwent CUBIC processing, multiplex immunofluorescence staining, and 3D imaging. Vascular density, surface area, vessel and muscle fiber diameters were compared between amyloid deposition and non-deposition regions. Ten additional specimens initially negative for AL amyloidosis were re-evaluated using the same procedure, suspected cases were verified by repeat histology and clinical follow-up.</p> Results <p>In amyloid deposition regions, vascular density and surface area were (0.14 ± 0.04) μm³ and (0.46 ± 0.11) μm², showing 50.0% and 33.3% reductions compared to non-deposition regions [(0.28 ± 0.06) μm³ and (0.69 ± 0.12) μm²]. Median vessel diameter in vessels with luminal amyloid deposition was 5.99&#xa0;μm (IQR: 5.07–7.39), 30.5% smaller than that in vessels without deposition [8.62&#xa0;μm (IQR: 7.26–11.03)]. Muscle fibers surrounded by amyloid deposits had a diameter of (3.62 ± 0.28)μm, slightly smaller than that of fibers without deposition [(3.70 ± 0.23)] μm. All differences were significant (<i>P</i> &lt; 0.05). Among the ten initially negative cases, five were reclassified as highly suspected and one was confirmed during follow-up.</p> Conclusions <p>CUBIC-based 3D imaging enables detailed visualization and quantification of amyloid-associated microstructural changes in tongue tissue, providing deeper insight into disease mechanisms and serving as a valuable complement to conventional histopathology for enhancing early diagnostic accuracy in AL amyloidosis.</p>