Surface plasmon resonance imaging (SPRi) is a unique microarray method for label-free and multiplexed bio-assays. However, it currently cannot be used to detect human serum samples due to its low sensitivity and poor specificity. A poly[oligo(ethylene glycol) methacrylate-co-glycidyl methacrylate] (POEGMA-co-GMA) brush was synthesized by surface-initiated atom transfer radical polymerization (SI-ATRP) and used as a unique supporting matrix for SPRi arrays to efficiently load probe proteins for high sensitivity while reducing nonspecific adsorptions for good selectivity. Results indicate that the polymer brush has a high protein loading capacity (1.8 protein monolayers), low non-specific protein adsorption (below the SPR detection limit), and high immobilization stability. Three model biomarkers, alpha-fetoprotein, carcinoembryonic antigen, and hepatitis B surface antigen were simultaneously detected in human serum samples by a SPRi chip for the first time, showing detection limits of 50, 20, and 100 ng mL(-1), respectively. This work demonstrates great potential for a SPRi biochip as a powerful label-free and high-throughput detection tool in clinical diagnosis and biological research. Since the SPR detection is limited by the sensing film thickness, this approach particularly offers a unique way to significantly improve the sensitivity in the SPR detecting thickness range.