Two-stage nanofiltration (TSNF) process was proposed for recovering high-value chemicals, including monophenols, cyclopentenones, glucose and acetic acid, from hydrolysates of lignocellulosic biomass (rice straw) through hydrothermal liquefaction (HTL). The separation performances of three single nanofiltration (NF) processes and three TSNF processes were studied. Results showed that at the first stage of (DL + DK) TSNF process, DL membrane had high glucose rejection of 97.12%, and lower acetic acid rejection as well as lower aromatics rejections than DK membrane. At the second stage, DK membrane had rather low acetic acid rejection of 5.04% to ensure acid separation from aromatics. Unlike glucose or acetic acid, aromatics were unable to be recovered into one fraction due to scattered rejections of different aromatic compounds on NF membrane. The (DL + DK) TSNF process was proved to be a feasible way to fractionate hydrolysates into three parts: glucose concentrate, monophenols and cyclopentenones concentrate, and acetic acid permeate.