The synthesis of tri‐(nitrilotriacetic acid) (NTA)‐end‐functionalized polystyrenes using an initiator containing tert‐butyl protected NTA moieties, by atom transfer radical polymerization (ATRP) of styrene is described. First, a suitable ATRP initiator is prepared and subsequently characterized by 1H and 13C NMR spectroscopy, gel‐permeation chromatography (GPC), and matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectroscopy. The structures of the tri‐NTA‐end‐functionalized polystyrenes (tri‐NTA‐PS) are confirmed by 1H and 13C NMR spectroscopy. Tri‐NTA‐PS itself produces self‐assembled spherical aggregates with ≈40–60 nm diameters in water/THF, whereas nickel‐complexed tri‐NTA‐PS produces spherical core–shell hybrid aggregates with ≈90–115 nm diameters with His‐tagged GFP in water/DMF (DMF 4 vol%), as confirmed by transmission electron microscopy and dynamic light scattering measurements.