There is an increasing demand on alternatives methods to animal testing. Numerous health parameters have been already studied using in vitro devices able to mimic the essential functions of the organs, being the real-time monitoring and response to stimuli their main limitations. Regarding the health of the gut, the short chain fatty acids, and particularly acetate, have emerged as key biomarkers to evaluate gut healthiness and disease development, although the number of acetate biosensors is still very low. This article presents a microbial biosensor based on fully biocompatible materials which is able to detect acetate in aerobic conditions in the range between 11 and 50 mM, and without compromising the viability and function of either bacteria (>90% viability) or mammalian cells (>80% viability). The detection mechanism is based on the metabolism of acetate by Escherichia coli bacteria immobilized on the transducer surface. Ferricyanide is used as a redox mediator to transfer electrons from the acetate metabolism in the bacterial cells to the transducer. High bacterial concentrations are immobilized in the transducer surface (109 cfu mL−1) by electrodeposition of conductive alginate hydrogels doped with reduced graphene oxide. The results show successful outcomes to exploit bacteria as a biosensing tool, based on the use of inkjet printed transducers, biocompatible materials and cell entrapment technologies. Display omitted •Escherichia coli uses acetate as carbon source in aerobic conditions.•Stable, biocompatible, conductive alginate hydrogel is electrodeposited on inkjet printed sensor's working electrode.•Immobilization of high bacterial concentrations of E. coli within alginate hydrogel.•Bacterial metabolism oxidizes acetate, enabling amperometric measurement of electron flow via ferricyanide reduction.•Successful proof-of-concept demonstration of bioelectrochemical acetate detection is performed.