In this work, we study how film thickness and chain packing density affect the protein-resistant properties of polymer brushes in complex media. Polymer brushes based on dual-functional poly(carboxybetaine acrylamide) (pCB) were prepared via surface-initiated photoiniferter-mediated polymerization. By adjusting UV radiation time and solvent polarity, pCB films with different thicknesses can be achieved and characterized using an ellipsometer. The packing density of pCB polymer chains is directly related to the swelling ratio of swollen to collapsed film thicknesses. Results showed that the dry film thickness alone, used often in the literature, is not sufficient to correlate with nonfouling properties and the chain packing density must be considered for the design of nonfouling surface coatings.