Cortisol, a famous stress biomarker, can be considered a potential predictor of cardiac diseases in humans. The presence of cortisol in saliva has encouraged researchers to design point-of-care devices for cortisol concentration in biological fluids. Here, human salivary cortisol was analyzed through a new non-invasive voltammetric aptasensor. Although cortisol is an electroactive compound, generally, the reduction in the current peak has been considered; however, this does not show a strong signal on a bare electrode surface, especially at low concentration levels. Hence, in this study, cortisol concentration was measured electrochemically and indirectly by monitoring the difference between electrochemical probe signals in the presence and absence of cortisol. A new polymeric nanocomposite of samarium molybdate flower-like nanoparticles decorated in poly(pyrrole) was electro-synthesized on the surface of a glassy carbon electrode. Then, reduced graphene oxide was cast on the surface. Finally, the cortisol aptamer was immobilized covalently on the reduced graphene oxide. This platform was used to increase the oxidation current peak of the ferricyanide solution as a probe as well as its electrocatalyst. The novel designed polymeric has the potential ability for effective immobilization of aptamers on the electrode surface without decreasing their biological activities. Additionally, it can enhance the probe electrochemical signal. The differential pulse voltammetric method (DPV) was applied as the detection technique. By optimizing the effective parameters, a determination range of 5.0 × 10−14–1.5 × 10−11 mol/L and a limit of detection of 4.5 × 10−14 mol/L were obtained. Selectivity of the proposed aptasensor relative to β-estradiol, progesterone and also prednisolone was studied as well. Finally, cortisol in a healthy human saliva sample was successfully analyzed by the proposed biosensors.