The two-photon excitation microscopy has become an important tool of noninvasive imaging due to the better penetration and relative harmlessness of the longer wavelength. However, the high photon flux in two-photon excitation can potentially lead to higher-order photobleaching within the focal volume. The relationship between the photobleaching rate and the excitation power for rhodamine 123 and rhodamine B in vivo and in vitro were measured. The coincidence of the results in vivo and in vitro demonstrated the correctness of the method. As expected, the photobleaching rate increased near-linearly with the excitation power for one-photon excitation. However, the two-photon photobleaching rate increased with high-order power (≥3.5) of excitation power, indicating the presence of high-order photon interaction in two-photon excitation microscopy. The same results are obtained by photobleaching experiments of the green fluorescence protein. As a consequence, the use of multi-photon excitation microscopy in the study may be limited by increased photobleaching.