Lignin is an abundant natural biopolymer found in plant cell walls. Lignin can come from tinctorial plants, whose residual biomass after dye extraction was typically discarded as waste. The main objective of this study was to extract lignin from the residual biomass of Reseda luteola L. using an organosolv process and to optimize the extraction conditions. The extracted lignin was characterized, and its potential applications as an antimicrobial, anti-oxidant, and anti-UV agent were investigated. Response surface methodology based on a Box-Behnken design was employed to optimize the lignin extraction conditions (organic acid concentration, material-to-liquid ratio, extraction time). The extracted lignin was comprehensively characterized using NMR, FTIR, XRD, SEM-EDX, TGA, DSC, and UV–Vis techniques. The optimal extraction conditions yielded a remarkably high lignin recovery of 62.41 % from the plant waste, which was rarely achieved for non-wood plants in previous works. The extracted lignin exhibited excellent thermal stability and radical scavenging anti-oxidant activity but no significant antimicrobial effects. Treating wool fabrics with lignin nanoparticles substantially enhanced UV protection from the “good” to “excellent” category based on the UPF rating. This sustainable valorization approach converted abundant tinctorial plant waste into high-purity lignin with promising anti-oxidant and UV-blocking properties suitable for various applications.