An amperometric nano-bioelectrode was designed that uniquely combined 1-pyrenebutyric acid units pi-pi stacked with carboxylated multiwalled carbon nanotubes on the surface of gold screen printed electrodes for covalent attachment of anti-insulin antibody or NAD⁺ dependent formaldehyde dehydrogenase. The designed enzyme bioelectrodes provided clinically relevant picomolar serum insulin measurements and detection of 6 ppb formaldehyde in 10-times diluted urine with a wide dynamic range, respectively. Fourier transform infrared, Raman, and electrochemical impedance spectroscopic characterizations confirmed the successful design of the bioelectrodes. Flow injection analysis provided lower detection limit and greater affinity for formaldehyde (apparent K_M 9.6 ppm) than stirred solution method (apparent K_M 19.9 ppm) due to better mass transport of the substrate. Moreover, selectivity assays revealed that the bioelectrode was selective toward formaldehyde with a moderate cross-reactivity for acetaldehyde (~ 25%) and negligible cross-reactivity toward propanaldehyde, acetone, methanol, and ethanol. The presented pyenyl carbon nanostructures-based bioelectrode design represents novelty and simplicity for enzyme-selective electrochemical detection of insulin hormone in serum and the small 30 Da formaldehyde marker present in urine. Similar strategy is broadly applicable for sensitive and selective electrochemical measurements of any other bio and small molecule markers present at ultra-low levels in complex clinical and environmental matrices.