Fluorescence resonance energy transfer (FRET) from a donor to multiple acceptors is an interesting subject. Numerous studies using theoretical models and simulations have focused on the excluded volume effect, which was not considered in Forster's first derivation. In this work, we first present the experimental results on the excluded volume effect by employing time-resolved FRET. Coumarin 334 (C334) was used as the energy donor whereas hemin and cytochrome c (cyt c) were used as the energy acceptors. The fluorescence intensity decays were measured for C334 surrounded by a number of acceptors in poly(acrylic acid). We have observed that the excluded volume effect is markedly pronounced with cyt c compared with hemin when the acceptor concentration is high (>5 mM). The results, which may be explicitly described by the relative molecular sizes of two acceptors, showed that the excluded volume effect should be considered in the interpretation of FRET data, especially when bulk chromophores are used.