In recent years, lignin-based nanomaterials have become increasingly relevant for researchers and producers of functional material applications due to their green and sustainable nature. However, there is still a challenge in controlling fabrication of lignin nanoparticles (LNPs). In the current study, we explored an environmentally friendly sequential hot water extraction with accelerated solvent extractor (ASE) to obtain a lignin-based fraction for the controllable production of LNPs. The lignin-based fractions are obtained from both Norway spruce heartwood (HW) and sapwood (SW) after sequential hot water extraction followed by separation with XAD 8 resin column and desorption with methanol (methanol fraction, MF). LNPs were successfully prepared from HWMF and SWMF with different physicochemical properties using acetonitrile/water binary solvent in an ultrasonic bath only within 1 min. The size of LNPs increased with the severity of wood ASE extraction, which is related to the reduction of β-O-4 bond, the increase of phenolic hydroxyl groups and the decrease of aliphatic hydroxyl groups in MF. However, no direct relationship between the size of LNPs and molar mass as well as carbohydrate content was found. The controllable preparation of LNPs was directly dependent on the ASE extraction conditions without complex chemical modification. This study presents a green method for controllable preparation of LNPs and provides a promising new value-added valorization pathway for lignin-based fractions (including lignin and lignin carbohydrate complex) from wood hot water extraction.