Micro- and nanosize lignin has recently gained interest due to improved properties compared to standard lignin available today. As the second most abundant biopolymer after cellulose, lignin is readily available but used for rather low-value applications. Applications for lignin in micro- to nanoscale, however, are ranging from improvement of mechanical properties of polymer nanocomposites, bactericidal and antioxidant properties and impregnations to hollow lignin drug carriers for hydrophobic and hydrophilic substances. This research represents an entire wheat straw biorefinery process chain and investigates the influence of Organosolv pretreatment conditions and precipitation parameters on particle size and morphology of nanolignin. A pretreatment temperature of 180 °C showed the best results in terms of morphology of the particles. Increasing addition flowrates of antisolvent to the Organosolv extract could decrease the particles size significantly to around 200 nm.