Surface-grafted polymer brushes have been long reported as a desired matrix for biosensing applications, owing to enhanced immobilization capacity and anti-fouling properties. However, simultaneous optimizing binding signal and minimizing non-specific adsorption using polymer matrix is still a challenge, due to the lack of systematic structural and composition optimization at molecular level. In this research, a series of block copolymer brushes with controlled hierarchical architectures, poly [oligo (ethylene glycol) methacrylate-b-glycidyl methacrylate] (POEMGA-b-PGMA) were prepared by two-stage surface-initiated polymerization. Surface plasmon resonance imaging (SPRi), as a real-time and label-free biosensing technology, was used as means to evaluate their detecting performances of bio-interactions. In contrast to one-layer poly (glycidyl methacrylate) (PGMA), 4.7-fold enhancement of signal-to-background (S/B) was achieved in bi-layer POEGMA-b-PGMA surface, by using rabbit IgG (R-IgG, in 10% serum) and a microarray of goat-anti-rabbit IgG (G-R-IgG) as the system of study. Interestingly, the binding signals and anti-fouling performances of the polymer brushes could be enhanced simultaneously by increasing the length of the monomer in the primary layer. Theoretically, it mainly owed to the formation of a densely packing protective primary layer against non-specific physical adsorption and a spatially optimized secondary layer of minimized steric hindrance that allowed optimum binding of analytes. (C) 2018 Published by Elsevier B. V.