Herein, we discussed the developments of silica-polysulfone based nanocomposite ultrafiltration membranes and their application in separation of protein, polysaccharide and humic substances. Two nanoparticles namely, mesoporous silica nanoparticles and nonporous silica nano-powder were impregnated on pure polysulfone matrix for preparation of nanocomposite membranes. Bovine serum albumin (BSA), sodium salt of alginic acid and humic acid sodium salt were used as model protein, polysaccharide and humic substances respectively. Membranes were characterized in terms of pure water permeability, separation of single uncharged solutes like polyethylene oxide (PEO), water contact angle and surface average roughness. The rejection of protein, polysaccharide and humic acid was also evaluated for all the membranes in order to get an idea whether these foulants can block the pores at some extent. Membranes containing porous silica were found more permeable and with nonporous silica are less permeable than the pure polysulfone membrane with similar solute rejection. Nanocomposite membranes were compared with pure polymer membranes for resistance to fouling by the model protein, polysaccharide and humic acid solutions. In general, the pure water flux recovery on flushing of water after fouling was more for nanocomposite membranes than the pure polysulfone membranes for all the foulants. BSA and humic acid sodium salt caused more irreversible fouling than sodium alginate in all the membranes. © 2015 Taylor and Francis Group, LLC.