Bioimaging technology has been broadly used in biomedicine, and the growth of multimodal imaging technology based on synergistic advantages can overcome the shortcomings of traditional single-modal bioimaging methods and attain high specificity and sensitivity in the fields of bioimaging and biosensing. The analysis of low-abundance microRNAs (miRNAs) in complex organisms is of high importance for early-stage diagnosis and clinical treatment of tumors. In our current study, a biosensing nanoplatform based on Tf-AuNCs and MnO2 nanosheets was developed for multimodal imaging of tumor cells. First, oxidizable MnO2 nanosheets provided a smart tool for use as nanocarriers and contrast agents for intracellular glutathione (GSH)-activated magnetic resonance imaging (MRI). Then, MnO2 nanosheets delivered Tf-AuNC-based biosensing nanoplatforms into cells through endocytosis. Endogenous GSH degraded MnO2 nanosheets into Mn2+, and the released functional nucleic acid probes can perform specific biosensing responses to miR-21 exhibiting multimodal imaging including two-photon near-infrared fluorescence imaging (TP-NIRFI), fluorescence lifetime imaging (FLIM), and MRI. Finally, the biosensing nanoplatform achieved satisfactory results in tumor cells and tissues by TP-NIRFI (300.0 μm penetration depth), FLIM (τ ≈ 50.0 ns), and MRI. Therefore, biosensing nanoplatforms based on Tf-AuNCs and MnO2 nanosheets show great potential for multimodal detection and imaging in tumor cells.