Circulating tumor cells (CTCs) have emerged as powerful biomarkers for diagnosis of prostate cancer. However, the effective identification and concurrently accurate imaging of CTCs for early screening of prostate cancer have been rarely explored. Herein, we reported a multifunctional gold nanoprobe-based thermophoretic assay for simultaneous specific distinguishing of prostate cancer CTCs and sensitive imaging of intracellular microRNA (miR-21), achieving the rapid and precise detection of prostate cancer. The multifunctional gold nanoprobe (GNP-DNA/Ab) was modified by two types of prostate-specific antibodies, anti-PSMA and anti-EpCAM, which could effectively recognize the targeting CTCs, and meanwhile linked double-stranded DNA for further visually imaging intracellular miR-21. Upon the specific internalization of GNP-DNA/Ab by PC-3 cells, target aberrant miR-21 could displace the signal strand to recover the fluorescence signal for sensitive detection at the single-cell level, achieving single PC-3 cell imaging benefiting from the thermophoresis-mediated signal amplification procedure. Taking advantage of the sensitive miR-21 imaging performance, GNP-DNA/Ab could be employed to discriminate the PC-3 and Jurkat cells because of the different expression levels of miR-21. Notably, PC-3 cells were efficiently recognized from white blood cells, exhibiting promising potential for the early diagnosis of prostate cancer. Furthermore, GNP-DNA/Ab possessed good biocompatibility and stability. Therefore, this work provides a great tool for aberrant miRNA-related detection and specific discrimination of CTCs, achieving the early and accurate diagnosis of prostate cancer.