A novel photochemical technological route for one-step functionalization of a graphene surface with an azide-modified DNA aptamer for biomarkers is developed. The methodology is demonstrated for the functionalization of a DNA aptamer for an N-terminal B-type natriuretic peptide (NT-proBNP) heart failure biomarker on the surface of a graphene channel within a system based on a liquid-gated graphene field effect transistor (GFET). The limit of detection (LOD) of the aptamer-functionalized sensor is 0.01 pg/mL with short response time (75 s) for clinically relevant concentrations of the cardiac biomarker, which could be of relevance for point-of-care (POC) applications. The novel methodology could be applicable for the development of different graphene-based biosensors for fast, stable, real-time, and highly sensitive detection of disease markers.