Heart failure has been acknowledged as a common disorder that leads to great morbidity and mortality. In heart failure and other cardiovascular disorders, effective biomarkers include cardiac natriuretic peptides, among which the most prominent one is B-type natriuretic peptide (BNP). The present study proposed the fabrication of a screen-printed working electrode (SPWE), with which a label-free and highly sensitive paper-based electrochemical immunosensor was successfully prepared. In addition, the SPWE was modified by amino functional graphene (NH2-G)/thionine (Thi)/gold nanoparticles (AuNPs) nanocomposites, which led to the immobilization of anti-BNP and an enhancement in the determination sensitivity. The linear range and limit of detection (LOD) were determined to be 0.05 to 30 ng/mL and 0.012 ng/mL, with a corresponding correlation coefficient of 0.996.