Remote activation of biomarker sensing holds a great promise of shifting the success of in vitro diagnostics to spatiotemporally controlled in vivo visualization of tumor, and in turn, imaging guided therapy. Herein, a "dual-key-one-lock" nanodevice was designed and built by assembling thermo-activatable probe of trimeric DNA hybrids into a mesoporous polydopamine nanoparticle-based multifunctional nanotransducer (probe host, fluorescence quencher, and photothermal conversion agent), enabling precisely switchable theranostic operations under the co-activation of exo/endogenous stimulations (near-infrared (NIR) light and microRNA (miRNA)). By this design, the NIR irradiation-induced local heat through the porous nanotransducer can be transferred to the DNA nanothermometer for triggering the exposure of the miRNA recognition segment, as well as the subsequent fluorescence activation by strand displacement reactions (SDR). A programmable application of short- (3 min) and long-duration (10 min) NIR irradiation was administered sequentially to induce a milder and a stronger hyperthermia, respectively, to activate the localized miRNA imaging, and in turn, tumor thermoablation under the fluorescence guidance in vivo. By reducing nonspecific activation, dual factor co-activatable nanodevices exhibited a high tumor-to-background ratio (TBR) value of 8.9, as well as a significantly lower (6–9-fold) normal tissue fluorescence as compared with those sensing miRNA solely. The in vivo results show that the tumors were significantly suppressed after the photothermal therapy with the assistance of the accurate miRNA diagnosis. This rationally integrated nanoplatform may pave a new avenue for advanced theranostic systems with high spatiotemporal precision by activatable designs.