Biosensing of NADH on bare electrodes has drawbacks such as high over-potential and poisoning during the oxidation reaction. To overcome this challenge a different approach has been undertaken by incorporating neutral red (NR) in Al doped ZnO (AZO) thin films using one-pot chemical bath deposition (CBD). The surface morphology of the films was hexagonal nanorods along the c-axis, perpendicular to the substrate. The thickness of the thin films were ranging from 400 to 3000 nm varying dependent on time of deposition (30 to 150 min). The average diameter of the nanorods was larger in the presence of neutral red (NR-AZO) with ~ 300 nm in contrast to its absence (AZO) with ~ 200 nm. The density of the packing of nanorods was dependent on the citrate concentration used during deposition. Control over the dopant concentration in the films was achieved by varying the area of Al foil used in the deposition solution. The selected area diffraction (SAED) and X-ray diffraction (XRD) indicated 002 plane of orientation in the nanorods. FTIR and FT-Raman analysis revealed conserved structure of NR and AZO. Chronoamperometric (CA) analysis showed a sensitivity of 0.45 μA cm− 2 mM− 1 and LoD of 22 μM within the range 0.075–4 mM of NADH. The biological sensing of NADH was validated by physical adsorption of NAD+ dependent-lactate dehydrogenase (LDH) on NR-AZO. CA showed sensitivity of 0.56 μA cm− 2 mM− 1 and LoD for lactate was 27 μM in the range of 0.1–1 mM of lactate. Further validation with real-time serum sample shows that LDH/NR-AZO correlates with the clinical values. The distinction in this study is that the organic mediator like neutral red has been incorporated into the grain structure of the ZnO thin film whereas other study with the mediators have only attempted surface functionalization. This article is part of a Special Issue entitled “Recent Advances in Bionanomaterials” Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader. © 2017 Elsevier B.V.