The membrane fouling mechanism used during the nanofiltration (NF) of chitin alkali effluent was investigated. Tests were carried out in three large-scale chitin-processing plants with three kinds of wastewater. An alkali resistant NF membrane with molecular weight cut-off of 250 Da was employed. The reflection coefficient (σ) and diffusion coefficient (Ps) of total proteins were deduced, assuming that the proteins were single entities in the feed. Viscosity and osmosis pressure were measured to evaluate their influences on the permeate flux. Furthermore, the fraction of the protein fouling was extracted and qualitatively analyzed by mass spectrometry. Results showed that the NF permeate flux of alkali wastewater with the highest protein concentration (4.00%) was the lowest, and that σ and penetration Ps decreased with protein content growth. Over 60% of the peptides in the permeate were hydrophobic, whereas 70% of the peptides in the adsorption cake were hydrophilic. Irreversible resistance was the predominant resistance during NF processing, and the fouling behaviour of hydrophilic fractions was dominant.