Fluorescent Au nanoclusters (NCs) are new excellent nanomaterials for biomedical applications but plagued by the problems of limited emission efficiency, unclear emission mechanism, and poor biological application ability. Herein, a novel strategy was developed to facilely synthesize poly(amidoamine) (PAMAM) dendrimer-hosted Au5 NCs (poly-Au5) with a high fluorescence quantum yield of 25%. Most importantly, a two-stage growth process of poly-Au5 was demonstrated through in situ time-course experiments. Stage I was a simultaneous self-nucleation and self-assembly with a rapid rate of fluorescence increase; stage II was a sole self-assembly after the end of reduction with a relatively slower rate of fluorescence increase but contributed 30% to the overall emission intensity of end products. In both stages, enhanced aurophilic interactions promoted the excited state relaxation dynamics; enhanced rigid structures reduced the level of nonradiative relaxition of excited states, and these two factors ensured high emission efficiency of poly-Au5. To further evidence the inference above, we successfully used PAMAM to realize the self-assembly of presynthesized, separated, and red-emitting Au-GSH NCs through electrostatic interaction between negative charges of carboxylic groups in Au-GSH NCs and positive charges of amine groups in PAMAM. As expected, the emission efficiency of Au-GSH NCs was obviously enhanced by PAMAM-mediated self-assembly. Moreover, the as-synthesized poly-Au5 assemblies exhibited excellent cell permeability and great biostability against various metal ions, high REDOX stress, and complex intracellular environments. By virtue of MnO4– as an intermediary agent, poly-Au5 was successfully used for sensitive and stable intracellular fluorescent imaging of endogenous GSH. This study lights up the emission origin of dendrimer-hosted Au NCs with strong emission and implies their huge applications in biomedical sensing and imaging.