A multi-functional nanohybrid (PyAnOH-Ag) with both a two-photon photothermal therapy (TP-PTT) effect and two-photon excited fluorescence (TPEF) imaging performance has been fabricated based on interfacial coordination interactions. The hybrid possesses a high two-photon absorption cross section (δTPA, 4638 GM) and detectable TPEF signals, which leads to excellent two-photon photothermal conversion. Upon irradiation at 840 nm, the temperature of the PyAnOH-Ag-PBS suspension reaches 42 °C in just 4 min, which results in 80% photothermal toxicity on HepG2 cells. The detectable TPEF signals can be used to monitor the cell ablation procedure. Moreover, PyAnOH-Ag exhibits a good phototherapeutic effect on tumor tissue of H22-modelled mice with almost 100% tumor growth inhibition under 840 nm irradiation for 10 min, which is superior to many reported photothermal agents. This strategy of TPEF guided TP-PTT agents can be potentially applied in a variety of therapeutic agents with monitoring ability.