Consecutive microcontact printing (μCP) has been developed to enable multiple functionalization of silicon surfaces, such as the immobilization of chiral ligands. The technique involves two subsequent printing steps using unstructured poly(methylsiloxane) stamps. The pattern is already defined on the substrate, consisting of etched channels. Hence, no precise alignment is needed between the two printing steps. A carboxylic acid group containing reagent was initially printed onto the silicon oxide surface and transformed to an anhydride. In the second printing step an ester bond was formed with the hydroxy-functionalized ligand. The formed molecular layers were evaluated by contact angle measurements, scanning electron microscopy (SEM) and electron spectroscopy for chemical analysis (ESCA), indicating that the consecutive μCP was successful. Initially, printing was performed on planar silicon surfaces but to realize a flow-through microfluidic device for high throughput screening a μCP technique was developed for etched channels. To verify the technique, hydrophobic valves consisting of octadecyltrichlorosilane were formed using μCP in deep reactive ion etched channels (50 μm wide and 50 μm deep). The printed hydrophobic patches were visualized by SEM and functioned well. Finally, the consecutive μCP technique was applied to immobilize the ligand in the channels. The channels were then sealed with a low-temperature bonding technique using an adhesive PDMS film, which does not destroy the printed ligand. In this study μCP is used in a novel manner. It enables a convenient method for performing complex surface modification of etched structures, which is a frequently appearing problem in biochemical microfluidic systems.