Measurement of ultra-low (e.g., parts-per-billion) levels of small-molecule markers in body fluids (e.g., serum, urine, saliva) involves a considerable challenge in view of designing assay strategies with sensitivity and selectivity. Herein we report for the first time an amperometric nano-bioelectrode design that uniquely combines 1-pyrenebutyric acid units pi-pi stacked with carboxylated multiwalled carbon nanotubes on the surface of gold screen printed electrodes for covalent attachment of NAD(+) dependent formaldehyde dehydrogenase (FDH). The designed enzyme bioelectrode offered 6 ppb formaldehyde detection in 10-times diluted urine with a wide dynamic range of 10 ppb to 10 ppm. Fourier transform infrared, Raman, and electrochemical impedance spectroscopic characterizations confirmed the successful design of the FDH bioelectrode. Flow injection analysis provided lower detection limit and greater affinity for formaldehyde (apparent KM 9.6 +/- 1.2 ppm) when compared with stirred solution method (apparent KM 19.9 +/- 4.6 ppm). Selectivity assays revealed that the bioelectrode was selective toward formaldehyde with a moderate crossreactivity for acetaldehyde (similar to 25%) and negligible cross-reactivity toward propanaldehyde, acetone, methanol, and ethanol. Formaldehyde is an indoor pollutant, and studies have indicated neurotoxic characteristics and systemic toxic effects of this compound upon chronic and high doses of exposure. Moreover, reported chromatography and mass spectrometry methods identified elevated urine formaldehyde levels in patients with bladder cancer, dementia, and early stages of cognitive impairments compared to healthy people. Results demonstrate that pyrenyl carbon nanostructures-based FDH bioelectrode design represents novelty and simplicity for enzyme-selective electrochemical quantitation of small 30 Da formaldehyde. Broader applicability of the presented approach for other small-molecule markers is feasible that requires only the design of appropriate marker-specific enzyme systems or receptor molecules. (C) 2017 Elsevier B.V. All rights reserved.