In this work, time-dependent growth of a poly(sulfobetaine methacrylate) (PSBMA) layer was realized on initiator-embedded and hydrophilic poly(N-vinylpyrrolidone)-containing (meth)acrylate resin, through subsurface-initiated atom transfer radical polymerization (SSI-ATRP). The antibiofouling performance of PSBMA graft layers with different thicknesses was investigated by testing the adhesion of different foulants, including nanoscale protein (bovine serum albumin, BSA), microscale bacteria (Gram-negative Escherichia coli (E. coli) and Gram-positive Bacillus subtilis (B. subtilis)) and diatom (Chaetoceros calcitrans). It was found that the optimal thickness for anti-adsorption of nanoscale protein is similar to 2 mu m at 1 h grafting, while that for microscale bacteria and diatom anti-adhesion is similar to 10 mu m at 4 h grafting. The difference in the optimal thickness for different foulants may result from the combination of effective hydration layer and surface topography. Regarding nanoscale protein, a relatively thin hydration layer may be able to provide effective steric repulsion to resist nanoscale protein adsorption. However, a relatively thick surface hydration layer associated with high steric barrier and large scale structure might be required to prevent microscale foulants from attaching. This study highlights the importance of graft layer thickness, which is related to the thickness of surface hydration layer, and topography in governing the antibiofouling performance against different foulants. [GRAPHICS] .