Atomic force spectroscopy (AFS) was used to measure interaction forces between the tip and nanostructured layers of poly(o-ethoxyaniline) (POEA) in pure water and CuSO4 solutions. When the tip approach and retraction were carried out at low speeds, POEA chains could be physisorbed onto the Si3N4 tip via nonspecific interactions. We conjecture that while detaching, POEA chains were stretched and the estimated chain lengths were consistent with the expected values from the measured POEA molecular weight. The effects from POEA doping could be investigated directly by performing AFS measurements in a liquid cell, with the POEA film exposed to liquids of distinct pH values. For pH ≥ 6.0, the force curves normally displayed an attractive region for POEA, but at lower pH values—where POEA is protonated—the repulsive double-layer forces dominated. Measurements in the liquid cell could be further exploited to investigate how the film morphology and the force curve are affected when impurities are deliberately introduced in the liquid. The shape of the force curves and the film morphology depended on the concentration of heavy metal in the liquid cell. AFS may therefore be used to study the interaction between film and analyte, with important implications for the understanding of mechanisms governing the sensing ability of taste sensors.