The synthesis of a two-photon fluorescence probe, {6-methoxy-2-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinolin-2-yl)benzo[d]thiazole} (MQH2O2), was achieved by incorporating 6-methoxybenzothiazol-2-hydroxyquinoline as the fluorescence moiety and borate ester as the H2O2 recognition element. Through the utilization of fluorescence spectroscopy and two-photon fluorescence spectroscopy, the ability of MQH2O2 to respond to H2O2 was evaluated. The results of the assessment reveal that the probe possesses a favorable H2O2 ratio response, with a ratio signal enhancement of roughly 25.4 times within 30 minutes and a detection limit as low as 38.6 nmol/L. Furthermore, the probe exhibits proficient two-photon properties, with a maximum two-photon fluorescence active cross-section of 150 GM. Finally, two-photon confocal imaging was utilized to conduct cell and brain tissue imaging, which demonstrated that the probe is capable of accomplishing in situ imaging analysis of oxidative stress caused by stroke.