An orange-red ［Ru(bpy)3］2+-DNA-Cu2+ composite film (bpy = 2,2′-bipyridine) was fabricated on an indium-tin (ITO) surface based on electrostatic interactions among ［Ru(bpy)3］2+, DNA and Cu2+ by using self-standing cast methods. The photoinduced electron transfer (PET) properties of the resultant composite film mediated by DNA were studied by means of steady-state and time-resolved fluorescence spectroscopy, UV-visible absorption spectroscopy, fluorescence microscopic imaging and scan electron microscopy. The ［Ru(bpy)3］2+-DNA-Cu2+ composite film with molar ratio of 10∶20∶1 shows an obvious absorption band (450 nm) and an intense emission peak (λem=595 nm), whose emission exhibits a single-exponential decay with τ=188.6 ns and is quenched by Cu2+ via DNA-mediated PET mechanism, indicating that the quenching constant is 6.94×103 L·mol-1 and quenching rate constant is 3.80×1010 L·mol-1·s-1. The increasing molar ratio of Cu2+ in composite films (10-fold) leads to an 11 nm blue-shift of the emission peak, which is dramatically weakened by Cu2+ via a static quenching mechanism. In addition, compared with the emission quenching of DNA-［Ru(bpy)2(tatp)］2+ (tatp=1,4,8,9-tetra-aza-triphenylene) tuned by Cu2+, which is present either in solutions or in composite films, Cu2+ only quenches the emission of ［Ru(bpy)3］2+ bound to DNA via an electrostatic interaction mode in composite films.