Although metal nanoclusters (MNCs) have shown great promise for the further development of photochemical techniques to be applied in diverse areas (e.g., photoelectronic devices, photochemical sensors, photocatalysts, and energy storage and conversion systems), the fundamental problem of their electron transfer behavior still remains unsolved. Herein, a driving force-dependent photoinduced electron transfer process of gold nanoclusters (AuNCs) is clarified for the first time from a rational-designed opposite-charged system. It was found that the electron transfer dynamic of carboxylated chitosan and dithiothreitol-commodified AuNCs (CC/DTT-AuNCs) can be satisfactorily described by the Marcus electron transfer theory. This proved model was applied to estimate the ultrafast charge separation process between CC/DTT-AuNCs and mitoxantrone, which was confirmed by fluorescence quenching and femtosecond transient absorption spectroscopy measurements. We envision that this work will open a new door for understanding the electron transfer behavior of MNCs and facilitate the design of advanced optoelectronic devices.