Patterning biomolecules at the micron or nano-scale presents a major challenge for the elaboration of integrated biochips. Among the different techniques that are emerging, Microcontact printing (µCP) appears as one of the most promising due to its simplicity and low cost. However, µCP exhibits a severe limitation because only one type of molecule can be deposited at a time using a poly(dimethyldiloxane) (PDMS) stamp. We present a new process called one-step-multiple-µCP (OSM-µCP) using a specific multilevel PDMS stamp which allows the patterning of two different molecules in one step. Our method based on the elastomeric properties of a PDMS stamp can print self aligned patterns of two different molecules by pressuring homogenously the top side of the stamp. The stamp levels inked by two different molecules contact the surface sequentially. OSM-µCP of two proteins, here bovin serum albumin BSA and streptavidin is demonstrated and this method can be applied to others couples of molecules. These results show that patterning biomolecules with OSM-µCP process opens new perspectives for soft lithography by enabling the fabrication of complex patterns of different molecules. .