Process conditions in the pulp and paper industry are closely linked to fiber quality but no analytical framework exists that simultaneously links the morphology and lignocellulose chemistry of fibers. Herein, optotracing is presented as a novel, non-destructive, fluorescence-based platform technology for assessment of lignocellulosic materials using intrinsic fluorescence from lignin combined with the external fluorophore Carbotrace 680, which provides a distinct optical signature when bound to cellulose. By applying fluorescence spectroscopy to microcrystalline cellulose/lignin model systems, distinct fluorescence signatures were assigned to each component. The signatures were validated on industrial never-dried and dried pulp, confirming that a quantitative relationship between lignocellulosic components can be generated in bulk samples. By translating the spectral settings to confocal microscopy and spectral imaging, cellulose and lignin were differentiated in individual fibers, signifying a role for optotracing in realizing their chemical relation at the microscopic level. Confocal microscopy also enabled simultaneous analysis of the morphology, physical dimensions, and lignocellulosic content, which when integrated into a spatio-chemical map revealed the lignocellulosic heterogeneity at sub-fiber resolution. By bridging chemical and morphological analyses across scales, optotracing platform technology represents a novel technology with a potential to revolutionize pulp analytics in research and industry alike.