In this work, we describe an approach of detecting biomarkers by Surface Plasmon Resonance imaging (SPRi) technique in real samples. Two C-Reactive Protein (CRP)-antibody immobilization methods were used: The first method was based on direct physisorption of CRP-antibody onto gold surface; the second one was based on oriented CRP-antibody with protein G intermediate layer. The two developed immunosensors were tested against CRP antigen in phosphate buffer saline solution with the SPRi technique. The response of the developed immunosensors was reproducible and stable. The detection limit of 10 pg·mL^-1 and 50 pg·mL^-1 CRP-antigen was observed with and without protein G respectively with this technique. Moreover, the developed SPRi immunosensor was used for CRP-antigen detection in human plasma. A detection limit of 5 ng·mL^-1 and 10 ng·mL^-1 was obtained with and without protein G respectively. These obtained results were compared to those obtained with QCM (Quartz Crystal Microbalance) and Enzyme-Linked Immunosorbent Assay (ELISA) techniques.