The purpose of this study was to compare the persistency of selected desensitizing agents after tooth brushing using fluorescence stereo zoom microscope. Forty 5 x 5 x 5-mm dentin discs were prepared from freshly extracted non-carious human first and second premolars and then were randomly divided into four groups (n = 10). Dentin desensitizing agents (All-Bond Universal, Gluma Desensitizer, and BisBlock) were applied with a fluorescent red dye (Rhodamine B). The persistency of the desensitizers was analyzed by fluorescence stereo zoom microscope (AxioZoom) over a period of eight weeks of tooth brushing. The mean florescence intensity (MFI; pixel) values were calculated for each specimen. The results were analyzed using Kruskal–Wallis, Mann–Whitney U, and Wilcoxon signed-rank tests with a significance threshold of p < 0.05. The BisBlock group showed statistically significant higher MFI values than the All-Bond Universal and Gluma groups in all time stages (baseline, 1st, 2 nd, 6 th, and 8 th weeks) (p < 0.05), whereas there were no statistically significant differences between the Gluma and All-Bond Universal groups (p > 0.05). However, the MFI values showed a reduction during the eighth week in all groups (p < 0.05). Tooth brushing caused various abrasion levels with the three desensitizing agents used in this study in proportion to time.