The fine control of the interfacial properties of functionalized graphene is a key point for its applications, especially in biosensing devices. We have here used an in-house developed technique to fabricate microsized patterned graphene via laser ablation and then we have functionalized the interface with poly-D-lysine, a bio-compatible polyelectrolyte normally used as a promoter for cell adhesion. Scanning Kelvin probe microscopy shows that a surface potential contrast appears at the patterned regions, with ablated regions of silicon substrate exhibiting higher surface potential than the surrounding background, whereas both levels have negative values. By subsequent coating with the poly-D-lysine it is possible to change stepwise the surface potential levels, while keeping the contrast at the patterned regions constant, up to neutralizing the initial negative values. With further dipping in a polyelectrolyte solution of opposite sign, such as polystyrene sulfonate, it is then possible to decrease the surface potential shifting it again back to negative values. The starting substrate chosen for graphene transfer allows deciding the sign of the surface potential contrast between two adjacent regions of the pattern.