Biofouling due to nonspecific proteins or cells on the material surfaces is a major challenge in a range of applications such as biosensors, medical devices, and implants. Even though poly(ethylene glycol) (PEG) has become the most widely used stealth material in medical and pharmaceutical products, the number of reported cases of PEG-triggered rare allergic responses continues to increase in the past decades. Herein, a new type of antifouling material poly(amine oxide) (PAO) has been evaluated as an alternative to overcome nonspecific foulant adsorption and impart comparable biocompatibility. Alkyl-substituted PAO containing diethyl, dibutyl, and dihexyl substituents are prepared, and their solution properties are studied. Photoreactive copolymers containing benzophenone as the photo-cross-linker are prepared by reversible addition-fragmentation chain-transfer polymerization and fully characterized by gel permeation chromatography and dynamic light scattering. Then, these water-soluble polymers are anchored onto a silicon wafer with the aid of UV irradiation. By evaluating the fouling resistance properties of these modified surfaces against various types of foulants, protein adsorption and bacterial attachment assays show that the cross-linked PAO-modified surface can efficiently inhibit biofouling. Furthermore, human blood cell adhesion experiments demonstrate that our PAO polymer could be used as a novel surface modifier for biomedical devices.