Abstract The development of enzymatic sensors for biological purposes such as biomedicine, pharmacy, food industry, and environmental toxicity requires the purification step of the enzyme. To prevent the loss of the enzyme activity, a new strategy is held in order to immobilize the bacteria. It will constitute the biological sensing element leading to a high operational stability and multiple adaptations to various conditions such as temperature, pH and ionic strength changes. In this work we describe the development of a urea biosensor by immobilizing Proteus mirabilis bacteria onto an insulator–semiconductor electrode on functionalized Fe3O4 nanoparticles (NPs), using cationic, Poly (allylamine hydrochloride) then anionic, Poly (sodium 4-styrenesulfonate) polyelectrolytes, BSA (serum bovin albumin), and glutaraldehyde as a cross-linking agent. The response of P. mirabilis to urea addition is evaluated in homogeneous and heterogeneous phases. Before the immobilization step, the activity of urease produced from the P. mirabilis bacteria was attempted using the ion ammonium selective electrodes (ISEs). Adhesion of the bacteria cells on IS electrodes have been studied using contact angle measurements. After immobilization of the bacteria, on the (Si/SiO2/Si3N4) and (Si/SiO2) substrates, the relationship between the evolution of the flat band potential ∆VFB and the urea concentration is found to be linear for values ranging from 10−2M to 10−5M.