The desorption forces of poly(4-vinylpyridine) (PVP) layer assemblies in different solvents were measured directly by using single-molecule force spectroscopy (SMFS). For pure methanol was used as the solvent, sawtoothlike force profiles were obtained, indicating that the detachment of polymer loops from the substrate was achieved. On addition of acetic acid into the pure methanol, the number of sawtoothlike force profiles decreased remarkably and more force curves with single rupture peak were obtained. However, in both cases a rupture force of approximately 180 pN was found frequently, which is thought to be the desorption force of an adsorbed PVP chain from a substrate tailored with amino groups.