In this study, cellulose nanocrystals (CNCs) were incorporated into a polyamide (PA) layer to prepare novel thin film composite (TFC) nanofiltration membranes (CNC-TFC-Ms). Scanning electron microscopy (SEM) results revealed differences in the surface morphologies of the CNC-TFC-Ms compared with that of a control membrane (TFC-control-M). The pore size distribution (PSD) results showed that the CNC-TFC-Ms exhibited smaller pore sizes and narrower PSD curves as the CNC content in the PA layer increased. Continuous potential measurements showed that the negativity of the surface charge of the CNC-TFC-Ms decreased as the CNC concentration increased. The CNC-TFC-Ms showed great rejection performance for divalent salts with rejection rates over 98.0% and 97.5% for Na2SO4 and MgSO4, respectively. Meanwhile, the filtration flux of salts was dramatically enhanced compared with that of TFC-control-M. Additionally, the permeate flux and rejection of NaCl were simultaneously enhanced as the CNC content increased, demonstrating that CNC-TFC-Ms can overcome the tradeoff limitation. In addition, the NaCl/Na2SO4 selectivity of the CNC-TFC-Ms reached as high as 60, suggesting their distinguished divalent/monovalent separation characteristics. The CNC-TFC-Ms exhibited greater dye removal for both anionic and cationic dyes due to their decreased negative charge and smaller pore size than the control membrane.