The invention relates to a method for separating and purifying the macromolecular color development matters in the outputted water of the Vc fermentation wastewater biochemical treatment. The Vc fermentation wastewater is the wastewater produced by the two-step fermentation method for producing Vc. The method comprises the following steps: (1) centrifugating the outputted water at a high speed, and filtering the outputted water through a microporous filter membrane so as to remove the suspended matters and solid particles in the outputted water; (2) determining the molecular weight distribution of the water sample through a gel filtration chromatography so as to determine the parameters of the intercepted molecular weight of an ultrafiltration membrane in the step (3); (3) according to the result of the step (2), selecting an ultrafiltration membrane with a proper intercepted molecular weight so as to carry out the ultrafiltration to obtain a penetrating fluid containing inorganic salts and micromolecular organisms and a trapped fluid containing the macromolecular color development matters; (4) adding deionized ultra pure water into the trapped fluid, and repeating the step (3); and (5) repeating the step (3) and step (4).