The large surface area/volume ratio and controllable surface conformation of electrospun nanofibers (ENFs) make them highly attractive in applications where a large surface area is desired, such as sensors and affinity membranes. In this study, nanocomposite-based ENFs were produced and immobilization of Anti-CRP was carried out for the non-invasive detection of C-reactive protein (CRP). Initially, the synthesis of graphene oxide (GO) was carried out and it was modified with magnetic nanoparticles (MNP, Fe₃O₄) and polydopamine (PDA). Catechol-containing and quinone-containing functional groups were created on the nanocomposite surface for the immobilization of Anti-CRP. Polystyrene (PS) solution was mixed with rGO-MNP-PDA nanocomposite and PS/rGO-MNP-PDA ENFs were produced with bead-free, smooth, and uniform. The surface of the screen-printed carbon electrode (SPCE) was covered with PS/rGO-MNP-PDA ENFs by using the electrospinning technique under the determined optimum conditions. Next, Anti-CRP immobilization was carried out and the biofunctional surface was created on the PS/rGO-MNP-PDA ENFs coated SPCE. Moreover, PS/rGO-PDA/Anti-CRP and PS/MNP-PDA/Anti-CRP immunosensors were also prepared and the effect of each component in the nanocomposite-based electrospun nanofiber (MNP, rGO) on the sensor response was investigated. The analytic performance of the developed PS/rGO-MNP-PDA/Anti-CRP, PS/rGO-PDA/Anti-CRP, and PS/MNP-PDA/Anti-CRP immunosensors were examined by performing electrochemical measurements in the presence of CRP. The linear detection range of PS/rGO-MNP-PDA/Anti-CRP immunosensor was found to be from 0.5 to 60 ng/mL and the limit of detection (LOD) was calculated as 0.33 ng/mL for CRP. The PS/rGO-MNP-PDA/Anti-CRP immunosensor also exhibited good repeatability with a low coefficient of variation.