The increasing use of graphene-based nanomaterials raises concerns about their potential environmental impact as ecotoxicants.This article examines the interaction and effects of graphene oxide (GO) and its amine-derived (GO-NH2) 2-D nanosheets on urease dynamics, a crucial enzyme in soil nutrient cycling, through spectroscopic, microscopic, and computational analyses.The findings reveal the distinct interaction mechanisms: GO formed ground - and excited-state complexes with urease; thus, fluorescence quenching was static and dynamic via hydrophobic interaction.In contrast, GO-NH2 nanosheets formed a ground-state complex by static quenching involving Van der Waals and hydrogen bonding.These interactions were energetically favored and altered tryptophan (Trp) residues′ microenvironment more than tyrosine (Tyr).The average exciton lifetime of urease in the presence of GO have decreased from 4.34 ns to 3.647 ns at the mere concentration of 7.11 μM, while for GO-NH2, no discernable change (4.16 ns) was observed even at the concentration of 16.75 μM.Primarily, the strong interaction of GO leads to the rearrangement of polypeptide structure, reduced α-helixes to 12 %, and increased β-sheets by six times.In contrast, with GO-NH2, α-helixes were reduced to 79 %, and β-sheets were increased by 1.8 times at the similar concentrationMol. docking revealed that neither GO nor GO-NH2 interacted with the active site of urease, and binding was primarily via hydrophobic, Van der Waals, and hydrogen bonding, concurrent with in-vitro incubation results.Pos. total energy (3.85 kJ mol-1) during mol. docking anal. of urease-GO interaction and inhibition in enzyme activity to 62 %, altered Vmax value (0.04606 ± 0.001 mM/s), without affecting the Km (4.78045 ± 0.12 mM) suggested that inhibition was noncompetitive and steric clashes due to disruptive binding might drove the inhibition.Meanwhile, neg. total internal energy (-3.97 kJ mol-1) during urease + GO-NH2 binding and enhanced enzymic activity by 32 % in-vitro without altering Vmax (0.063091 ± 0.003 mM/s) and Km (4.953943 ± 0.2 mM) values suggested that urease′s binding site was well-complemented by GO-NH2 nanosheets, serving as a redox-mediator and radical-quencher.