Manipulating micro-objects plays a crucial role in a wide range of fundamental and applied research works. Here, we propose an original strategy based on the chemical modification of a substrate by hydrogen-bonded films elaborated by layer-by-layer (LbL) assemblies of poly­(ethylene glycol) (PEG) and poly­(acrylic acid) (PAA). First, the influence of the polymer molecular weight on the growth of the PEG/PAA multilayer was evaluated. Optical reflectometry analysis used to follow in situ the film buildup revealed a strong dependence of the deposited amount of polymer on the ratio of monomer units of each polymer (n PAA/n PEG). Then, colloidal probe atomic force microscopy (AFM) microscopy was carried out in an aqueous medium to monitor the adhesion forces of multilayer surfaces composed of N polymer layers. Pull-off forces were converted using the Johnson–Kendall–Roberts (JKR) model to access the thermodynamic work of adhesion. Results indicated that PEG/PAA multilayer films exhibit weak adhesion forces that are sensitive to the number of deposited polymer layers at pH 2. In addition, a progressive increase of the solution pH reduced the adhesion due to the destruction of the hydrogen-bonded multilayer film. To simulate the capture and the release of a micro-object, borosilicate particles acting as spherical micro-objects were adsorbed onto a PEG/PAA film. Once again, an increase of the solution pH led to desorption of particles, as shown by optical microscopy. Finally, an AFM tip functionalized by a PEG/PAA multilayer was used to achieve successful micromanipulation operations (capture and release) of a 10 μm diameter borosilicate sphere in an aqueous solution.