Heart failure (HF) is a clinical hallmark of cardiovascular syndrome leading to substantial morbidity, repeated hospitalization, and enormous healthcare expenditure. As elderly populations continue to increase, biomarker-guided diagnosis and treatment for age-related cardiac decline have become essential. Accumulating evidence demonstrates that N-terminal proBNP (NT-proBNP) can provide a unique window into the diagnosis and risk stratification with HF. Herein, a simple yet robust aptasensor is developed for on-site recognizing the NT-proBNP by its targeting aptamer, thus achieving the accurate diagnosis of HF. This aptasensing system is prepared by absorbing the fluorophore-labeled aptamer strand onto the graphene oxide (GO), leading to efficient quenching without possible off-site signal leakage. The aptamer strand can specifically identify target NT-proBNP molecules via a versatile conformational transformation, resulting in the desorption of the aptamer-NT-proBNP complexes from GO and re-generation fluorescence signal, thus allowing sensitive detection of NT-proBNP in 37 clinical blood samples. Taking advantage of the high specificity of aptamer-guaranteed recognitions of NT-proBNP, this aptasensor system readily achieves better diagnostic performance for HF than commercially adopted chemiluminescence immunoassay (Siemens, CLIA) in hospitals in terms of accuracy (89.2% vs 83.8%), specificity (89.5% vs 84.2%), and positive predictive value (88.9% vs 83.3%). This work provides a stable option for the diagnosis and treatment of elderly HF-related diseases in clinics.