The invention discloses a method for separating and extracting N-acetyl-D-glucosamine and D-glucosamine from ammonia sugar fermentation liquor. The method includes the steps of microfiltration, ultrafiltration, nanofiltration and the like, the fermentation liquor is filtered through a microfiltration membrane with the aperture of 0.1-10 micrometers to remove impurity particles and microbial thalli, protein, nucleic acid, colloidal particle large molecules and other impurities are removed through filtering by means of an ultrafiltration membrane with the aperture of 0.001-0.1 micrometer, and pigment, polypeptide, nucleotide and other small-molecular impurities are removed through a reverse osmosis system; N-acetyl-D-glucosamine is crystallized and separated from the fermentation filtrate containing N-acetyl-D-glucosamine and glucosamine. The method includes the steps of fermentation filtrate heating and concentration, seed crystal addition, temperature-control crystallization, N-acetyl-D-glucosamine crystal collection, residual glucosamine recycling and the like. The method is low in cost and does not influence the structure of N-acetyl-D-glucosamine, and the obtained N-acetyl-D-glucosamine is high in purity.