Cholesterol (C27H46O stated as M) is used as a model of bio‐organic contamination, because this compound is in almost every living organism, and its photocatalytic degradation on titanium oxide (TiO2) surface with UV exposure is investigated. Secondary ion mass spectrometry as a surface‐sensitive technique is suited to study this degradation process with focus on intermediates. A fragment of M–OH represents the intact molecule of cholesterol and provides its relative concentration on the surface. The intensity of M–OH decreases after 24‐h UV irradiation, and the level of degradation is 89% with the pseudo‐first kinetic constant of 0.0207 min−1 within 2 h. A fragment of MO–H represents an intermediate as one of the cholesterol oxidation products. The irradiation from a bottom in comparison with a top reveals the differences in the mechanism of the intermediate formation through the intensity and the kinetics with values of factor of 1/3 and 30 min, respectively. The roles of electrons and holes, primarily generated in TiO2 by UV, and also of superoxide anion radical and hydroxyl radical, as the secondary reactive species, are discussed to illustrate the bottom/top mechanisms. Copyright © 2016 John Wiley & Sons, Ltd.