A method to tune the thickness and number of available functional groups of micropatterned layers of a polycation stamped onto weak polyelectrolyte multilayers has been developed. Specifically, poly(allylamine hydrochloride) (PAH) was transferred using polymer-on-polymer stamping to create chemically patterned regions on weak polyelectrolyte multilayer films of poly(acrylic acid) (P-AA) and PAH, presenting PAA as the outermost layer. Polyelectrolyte multilayer platforms ranging in molecular-level architecture from tight ionically cross-linked to thick and loopy were created by using specific combinations of polymer solution pHs during the multilayer assembly. It was found that varying the pH of the stamping ink significantly affects the thickness of the resulting transferred layer and the number of functional groups available for further chemistry. As the weak polyamine in the ink solution becomes less charged at higher pH, the thickness of the stamped pattern increases. The PAH stamped regions were also capable of directing specific chemistry on the multilayer surface through the binding of the fluorescent marker dansyl chloride. In addition, analysis of the fluorescent images indicates that more amine groups react as the patterned layer becomes thicker. The stamped features were stable even after extended rinses in buffer solution, making the patterned multilayer films suitable for biological applications. These micropatterned films are excellent candidates for biosensors due to the functionalities of both the underlying surface and the stamped polymers.