A novel immunoassay (IA) has been developed for human C-reactive protein (CRP), an important biomarker and tissue preserving factor for infection and inflammation. Graphene nanoplatelets (GNP) and 3-aminopropyltriethoxysilane (APTES) were admixed and covalently attached to a polystyrene based-microtiter plate (MTP), pretreated with KOH. The resulting surface served as a stable layer for the covalent attachment of the anti-human CRP antibody. The IA procedure was based on the one-step kinetics-based sandwich IA employing a minimum number of process steps, whereas the enzymatic reaction solution was monitored by a smartphone-based colorimetric reader. With a limit of detection and a limit of quantification of 0.07 ng mL−1 and 0.9 ng mL−1, it precisely detected CRP spiked in diluted human whole blood and plasma as well as the CRP levels in clinical plasma samples. The results obtained for “real-world” patient samples agreed well with those of the conventional immunosorbent assay and the clinically-accredited analyzer-based IA. The antibody-bound GNP-functionalized MTPs retained its original activity after 6 weeks of storage in 0.1 M PBS, pH 7.4 at 4 °C.