The production of lignocellulosic-based materials requires leveraging alternative biomasses such as agricultural residues. This study investigates Lupinus angustifolius as a renewable feedstock for lignin-containing microfibrillated cellulose (L-MFC) and explores the role of lignin and hemicellulose in producing lupin-derived L-MFC films. By utilizing mild alkaline pre-treatments (0.5 or 1.5 M NaOH at 90 or 140 °C) and avoiding extensive delignification, four lignocellulose systems with varying lignin and hemicellulose content are generated. To produce L-MFC, these systems were further subjected to a defibrillation process, including TEMPO oxidation and mechanical homogenization. TEMPO oxidation partly removed the lignin while preserving hemicellulose, leading to altered biopolymer ratios. Confocal fluorescence microscopy combined with optotracing enabled real-time visualization of the defibrillation process and spatial biopolymer mapping. Complementary excitation-emission matrix (EEM) spectroscopy provided fluorescence fingerprints for tracking molecular-level changes during processing. The resulting lupin-derived L-MFCs were processed into self-standing films using Rapid Köthen drying. Films with higher residual lignin displayed increased hydrophobicity and mechanical strength with a tensile index of up to 110 Nm/g and a Young's modulus of up to 9700 MPa. This study demonstrates that partial preservation of native lignocellulose architecture offers a viable pathway for sustainable lupin-derived L-MFC film production.