Abstract [en]The macroalga Ulva fenestrata plays a key role in marine ecosystems and has increasing potential in aquaculture. However, its three-dimensional tissue architecture remains underexplored. This study applies multimodal fluorescence microscopy combined with optotracing to spatially map biopolymers and structural features in native Ulva tissue. Using Carbotrace 680, cellulose was localized in situ within the cell walls, while oligo/polyaromatic compounds were visualized across multiple scaffold layers via autofluorescence. Lambda scanning validated the fluorescence detection settings for cellulose (Exitation wavelength (Ex.) 561 nm, Emission wavelength (Em.) 570–631 nm), oligo/polyaromatics (Ex. 405 nm, Em. 408–505 nm), and chlorophyll (Ex. 639 nm, Em. 649–693 nm). Spatially resolved biopolymer anatomy maps were generated for blade and rhizoidal tissues, and 3D tissue models were constructed. The outermost blade layer exhibited a sandwich-like architecture, and a previously undescribed median layer was identified separating the two cell layers. This layer was >11 times thicker in rhizoidal tissue than in blade tissue, comprising 56 % and 7 % of the total thickness, respectively. Spectral differences in rhizoidal cells indicated cellular heterogeneity. Collectively, the observed biopolymer and architectural differences may reflect tissue-specific functional specialization of the macroalga. This imaging-based approach provides new perspectives on algal biology and supports the multisectoral valorization of Ulva.