Fluorescence molecular tomography (FMT) imaging can be used to determine the location, size, and biodistribution of fluorophore biomarkers inside tissues. Yet when using FMT in the reflectance geometry it is challenging to accurately localize fluorophores. A depth perturbation method is proposed to determine the centroid of fluorophore inside a tissue-like medium. Through superposition of a known thin optical phantom onto the medium surface, the fluorophore depth is deliberately perturbed and signal localization is improved in a stable way. We hypothesize that the fluorophore centroid can be better localized through use of this fluorescent intensity variation resulting from the depth perturbation. This hypothesis was tested in tissue-like phantoms. The results show that a small-size fluorophore inclusion ( volume, depth up to 4.8 mm) can be localized by the method with an error of 0.2 to 0.3 mm. The method is also proven to be capable of handling multiple fluorescent inclusion conditions with the assistance of other strategies. Additionally, our further studies showed that the method’s performance in the presence of background fluorophores indicated that the small inclusion could be located at a 1.8 (3.8) mm depth with accurate localization only when its concentration was not (100) times the background level.