In order to reveal the influences of fouling layers formed by different organic matters on gypsum scaling behaviors on the surface of nanofiltration membranes, bovine serum albumin (BSA), humic acid (HA) and alginate (SA) were chosen as typical organic foulants to contaminate the composite polyamide nanofiltration membrane, respectively. After that, the gypsum scaling experiment was conducted. The gypsum crystal morphologies of different fouling conditions were observed by scanning electron microscopy (SEM). The interaction forces of membrane-gypsum and gypsum-gypsum in different membrane fouling conditions were investigated by atomic force microscope (AFM) combined with self-made gypsum probe. The results indicated that, compared with the virgin membrane condition, the adsorption of organic foulants on nanofiltration membrane had changed the membrane surface properties and influenced gypsum crystal nucleation mechanism. The degree of gypsum scaling in three organic fouling conditions decreased in the order of SA>HA>BSA. These phenomena were likely due to the interactions between SA and Ca²⁺ which could shorten the nucleation time of gypsum crystals, enlarge the crystals size and result in the fastest membrane flux decrease. The complexation between HA and Ca²⁺ increased the thickness and compaction of scaling layer, leading to a relatively serious membrane flux decline. Besides, the weak Ca²⁺ combination ability and particular heart-shape molecular structure of BSA led to a less degree of gypsum scaling compared to the other two kinds of organic conditions.