AbstractZwitterionic polymers are promising non‐fouling materials widely used for surface modification. Here, we report the synthesis of zwitterionic polyaspartamides bearing carboxybetaine and sulfobetaine prepared via combining the ring‐opening reaction of polysuccinimide with 3‐(dimethylamino)‐1‐propylamine and the reaction of the formed tertiary amine side groups with 1,3‐propanesultone and 4‐bromobutyric acid, respectively. Both sulfobetaine and carboxybetaine polyaspartamides have cationic and anionic charged groups on the same repeat unit, but they differ in negatively charged groups. The chemical structures of the synthesized polymers were confirmed by 1H NMR and FT‐IR spectroscopies. The zwitterionic property of polyaspartamides were characterized by zeta potential measurements using wide range of pH. The surface properties mainly anti‐protein adsorption abilities and hydrophilicity were tested by surface plasmon resonance analysis, fluorescence microscopy, and contact angle measurements. Importantly, sulfobetaine polyaspartamides demonstrated better both antifouling performance and ability to form more hydrophilic surfaces than carboxybetaine polyaspartamide. Two zwitterionic carboxybetaine and sulfobetaine nanogels were prepared by horseradish peroxidase‐mediated crosslinking in an inverse miniemulsion. Results from dynamic light scattering and transmission electron microscopy showed the formation of nanosized spherical particles. Additionally, sulfobetaine and carboxybetaine polyaspartamides had remarkable biocompatibilities, indicating their promise as non‐fouling materials for biomedical applications.