Wearable devices seem to have great potential that could result in a revolutionary non-clinical approach to continuous health monitoring. Stretchable biosensors in this regard have shown promising potential for their use in wearable devices owing to their ability to act as an interface between physiological environment and electronically generated signals. Such stretchable biosensors should be made of nontoxic, non-irritant, conductive, and flexible materials. Recently conductive hydrogel-based stretchable biosensors are gaining huge interest due to their potential for immobilization of analytes, biocompatibility, water retention ability and stretchability like skin, in addition to their tuneable physical and electrochemical properties. Nano-enabled hydrogels have further demonstrated enhanced sensitivity and selectivity in biosensors. To this end, this study focused on development of novel bio-nanocomposite hydrogel substrate as wearable biosensing devices and its systematic investigation of microstructure and electrochemical behaviour. Thermally exfoliated graphene oxide (TEGO) was synthesized and incorporated as a nanofiller within the hybrid biopolymer matrix consisting of polyvinyl alcohol (PVA) and conductive polyaniline (PANI). The synthesised TEGO was found to have intercalated nanostructure, and were dispersed uniformly within the hybrid matrix, as confirmed by FE-SEM investigation, anticipated to impart increased interfacial adhesion. Physical crosslinking of polymer networks was achieved by repetitive freeze thaw cycle. The flexible and unique hierarchical structure developed by incorporating TEGO within the conductive biopolymer system also enhanced the electrical conductivity in a significant way as indicated by the electrochemical impedance spectroscopy (EIS). This study, therefore, could establish the promising potential of developed bio-nanocomposite hydrogels in wearable biosensing devices. (c) 2022 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the Second International Conference on Engineering Materials, Metallurgy and Manufacturing.