A novel immunosensing strategy based on surface-initiated atom-transfer radical polymerization (SI-ATRP) in combination with electrochemical detection is proposed. Specifically, 4-acetoxystyrene (AS) has been chosen as a monomer for ATRP due to its ability to provide acetoxyl groups, which can be converted into phenolic hydroxyl groups for electrochemical detection in the presence of tyrosinase. A controlled radical polymerization reaction of 4-acetoxystyrene at 60 °C was triggered after immobilization of initiator molecules on an electrode surface. The growth of long-chain polymeric materials increased the concentration of phenolic hydroxyl groups, which in turn significantly enhanced the electrochemical signal output. Polymerization conditions, such as temperature and duration, monomer concentration, and the catalyst/monomer ratio have been optimized. The in situ surface-initiated ATRP was confirmed by scanning electron microscope (SEM) images and X-ray photoelectron spectroscopy (XPS) analysis. Cyclic voltammetric investigation revealed a pair of well-defined oxidation and reduction peaks at 0.232 and 0.055 V, which corresponded to the redox behavior of catechol/o-quinone on the electrode surface. The proposed approach has been successfully extended to immune recognition. A detection limit of 0.3 ng mL−1 for rabbit immunoglobulin G (IgG) as a model antigen has been achieved. Despite the limited availability of the IgG antibody, this technology might also be expanded to the detection of other proteins and DNA.