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• An excellent bi-color probe named PEG@ReSe 2 nanosheets was prepared. • The nanoprobe exhibited strong two-photon features under a low power excitation. • The nanoprobe showed strong photostability, low cytotoxicity, enabling to perform in vivo bioimaging in cancer cells. Recently two-dimensional (2D) nanomaterials-mediated photothermal therapy (PTT) has become an important tool for treating cancer. However, visually monitoring the localization of nanoagents in cancer cells remains a key challenge. Herein, exploring high photostability, and high biocompatibility probes for monitoring PTT are still highly desirable. In this study, a novel optical probe named PEG@ReSe 2 nanosheets (polyethylene glycol, PEG; rhenium diselenide, ReSe 2) was prepared and employed to perform in vivo two-photon imaging for monitoring PTT in esophageal cancer cells. Under the illumination of a near-infrared (NIR) femtosecond laser at 820 nm with the output power as low as 5 mW, the PEG@ReSe 2 nanosheets showed significant two-photon features, producing a significant photoluminescence emission band at 637 nm. In addition, the prepared PEG@ReSe 2 nanosheets exhibited high resistance to photobleaching. Also, the PEG@ReSe 2 nanosheets showed low cytotoxicity for cancer cells, providing a high viability of more than 90%. More importantly, the PEG@ReSe 2 nanosheets could enter cancer cells and work as a two-photon probe for visually displaying the PTT process. After 12 h incubation, the PEG@ReSe 2 nanosheets produced strong two-photon signals in KYSE150 cancer cells, enabling them to perform in vivo bi-channel bioimaging. In light of its photothermal effects, the PEG@ReSe 2 nanosheets also could serve as a sensor for monitoring PTT under low-power infrared laser irradiation. This study suggests that, the PEG@ReSe 2 nanosheets could serve as a superior two-photon biosensor for monitoring the PTT process in cancer nanomedicine. [ABSTRACT FROM AUTHOR]