Therefore, the FTIR examination confirms the binding of ammonium ions to the carbon atoms in the graphene structure. The binding was further validated by Raman spectroscopic analysis where the control and treated samples were studied for the changes in the edge defects, disorder and functional groups. The plots demonstrate the significant changes in the graphene layers upon NH4 + incorporation, which was inferred by increasing AD/AG. The obtained results suggested the formation of C-N bonding and incorporation of nitrogen atom in the hexagonal lattice thereby leading to functionalization. The FTIR and Raman behaviour of the heat treated samples revealed that changes, observed in the ammonium treated graphene samples, were found to be absent in the heated samples, which marks the reproducibility of the bioprepared graphene as ammonium ions sensor. Hence, the biopreparation of graphene layers can be useful for real life application in ammonium ions sensing in human urine to detect the changes due to kidney dysfunctionality.