MiRNA-targeted therapy has become a hot topic in current cancer research. The key to this treatment strategy is to clarify the specific role of miRNA in cancer. However, the roles of some miRNAs acting as oncogenic or tumor suppressors are still controversial, which are influenced by different tumor types, even in the same cancer type. Hence, we designed a novel fluorescent nanoprobe based on polydopamine nanoparticles (PDA NPs) for simultaneously detecting caspase-3 and miRNA-34a within living cells. The specific role of miRNA-34a in different cancer cells could be further identified by studying the expression alterations of caspase-3 and miRNA-34a. Confocal imaging indicated that miRNA-34a indeed acted as a tumor suppressor in anticancer drug-treated MCF-7 and HeLa cells, where the effect of miRNA-34a remains controversial. The designed nanoprobe can offer a promising approach to ascertain the oncogenic or tumor-suppressing role of miRNA in different cancer cells with a simple visualization method, which has valuable implications for exploring the practicability of precision therapy focused on miRNA and evaluating the efficacy of new miRNA-targeted anticancer medications.