The rational design of biomaterials with antifouling properties still remains a challenge, although this is important for many bench-to-bedside applications for biomedical implants, drug delivery carriers, and marine coatings. Herein, we synthesized and characterized poly(N-acryloylglycinamide) (polyNAGA) and then grafted poly(NAGA) onto Au substrate to form polymer brushes with well-controlled film stability, wettability, and thickness using surface-initiated atom transfer radical polymerization (SI-ATRP). The NAGA monomer integrates two hydrophilic amides on the side chain to enhance surface hydration, which is thought as a critical contributor to its antifouling property. The antifouling performances of poly(NAGA) brushes of different film thicknesses were then rigorously assessed and compared using protein adsorption assay from undiluted blood serum and plasma, cell-adhesive assay, and bacterial assay. The resulting poly(NAGA) brushes with a film thickness of 25-35 nm exhibited excellent in vitro antifouling ability to prevent unwanted protein adsorption (<0.3 ng/cm) and bacterial and cell attachments up to 3 days. Molecular dynamics (MD) simulations further showed that two hydrophilic amide groups can interact with water molecules strongly to form a strong hydration layer via coordinated hydrogen bonds. This confirms a positive correlation between antifouling property and surface hydration. In line with a series of polyacrylamides and polyacrylates as antifouling materials synthesized in our lab, we propose that small structural changes in the pendent groups of polymers could largely improve the antifouling capacity, which may be used as a general design rule for developing next-generation antifouling materials.