The nanoparticles of commercially available pyruvate oxidase (POx) from Aerococcus species were prepared by desolvation method, which were then characterized and covalently immobilized onto gold electrode (AuE) to construct an improved model of amperometric pyruvate biosensor. The POxNPs/Au electrode was analyzed morphologically by scanning electron microscopy (SEM). On the other hand, cyclic voltammetry studies (CV) and electrochemical impedance spectroscopy (EIS) helped in deciphering the electrochemical properties of the electrode at different stages of construction. The biosensor showed optimum response within 7.5 s, at a potential of 0.28 V, pH 5.5 and 35 degrees C. A linear relationship was observed between biosensor response i.e. current (mu A) and pyruvate concentration in the range, 0.01 mu M - 5000 mu M, with a lower detection limit of 0.67 mu M. The analytical recovery of added pyruvate in sera was 99.0% and 99.5% within and between batch coefficient of variation (CV) were 0.045% and 0.040% respectively. The working electrode displayed an excellent correlation coefficient (R-2 = 0.99%) between levels of pyruvate in sera, as detected by the standard spectrophotometric method and the present biosensor. The biosensor was utilized for detection of total pyruvate level in sera of apparently healthy individuals and patients suffering from cardiogenic stress, more specifically cardiac failure. The activity of the biosensor deteriorated by 25%, after its regular use over a period of 240 days, while being stored dry at 4 degrees C.