In order to investigate the roles of active molecules during the bioinspired synthesis process of gold nanoparticles by Cacumen platycladi leaf extract, flash column chromatography was employed to separate the Cacumen platycladi leaf extract according to the polarity differences. Fourier transform infrared spectroscopy and high-performance liquid chromatography equipped with a photodiode array detector were adopted to identify the category of the isolated samples. The results have demonstrated that components in the Cacumen platycladi leaf extract can be sorted into three categories: sugars (the Sloading and S0), polyphenols (S1 similar to S5), and flavonoids (S6 similar to S8). The characterizations of gold nanoparticles, produced by each isolated sample with chloroauric acid, indicate the strong-to-weak sequence of reducing power is polyphenols, flavonoids, and sugars. Sugars have no protection strength for the biosynthesis of gold nanoparticles, polyphenols involved in isotropous stabilization, while flavonoids are responsible for the anisotropic growth.