High‐dimensional imaging mass cytometry (IMC) enables simultaneous quantification of over 35 biomarkers on one tissue section. However, its limited resolution and ultralow acquisition speed remain major issues for general clinical application. Meanwhile, conventional immunofluorescence microscopy (IFM) allows sub‐micrometer resolution and rapid identification of the region of interest (ROI), but only operates with low multiplicity. Herein, a series of lanthanide‐doped blue‐, green‐, and red‐fluorescent carbon nanodots (namely, B‐Cdots(Ln1), G‐Cdots(Ln2), and R‐Cdots(Ln3)) as fluorescence and mass dual‐modal tags are developed. Coupled with aptamers, B‐Cdots(159Tb)‐A10‐3.2, G‐Cdots(165Ho)‐AS1411, and R‐Cdots(169Tm)‐SYL3C dual‐functional aptamer probes, which are then multiplexed with commercially available Maxpar metal‐tagged antibodies for analyzing clinical formalin‐fixed, paraffin‐embedded (FFPE) prostatic adenocarcinoma (PaC) tissue, are further synthesized. The rapid identification of ROI with IFM using fluorescence signals and subsequent multiplexed detection of in situ ROI with IMC using the same tissue section is demonstrated. Dual‐modal probes save up to 90% IMC blind scanning time for a standard 3.5 mm × 3.5 mm overall image. Meanwhile, the IFM provides refined details and topological spatial distributions for the functional proteins at optical resolution, which compensates for the low resolution of the IMC imaging. The MC‐Cdots(Ln)‐aptamers serve as dual‐modal probes that realize the rapid identification of region of interest (ROI) with immunofluorescence microscopy using fluorescence signal, and then multiplexed detection of in situ ROI with imaging mass cytometry on the same tissue section.