Fluorescence resonance energy transfer (FRET) is non-radiation energy transfer that occurs between a donor (D) molecule in an excited state and an acceptor (A) molecule in a ground state by dipole-dipole interactions. The efficiency of FRET is dependent on the extent of spectral overlap between the donor photoluminescence peak and the absorption spectrum of acceptor, the quantum yield of the donor, and the distance between the donor and acceptor molecules. Currently, FRET is commonly used for determining the metal ion, analyzing the protein, biological molecular fluorescence probe, etc. In this study, the FRET between CdTe quantum dots (QDs) with different sizes and Rhodamine B (RhB) in aqueous solution is investigated by using the time-resolved fluorescence test system under two-photon excitation. In this two-photon FRET aqueous system, QD is used as donor while RhB as acceptor. The time resolved two-photon photoluminescence and fluorescence lifetime measurements are performed for analyzing the two-photon-excited luminescence by using a titanium sapphire femtosecond laser with a wavelength of 800 nm, pulse width of 130 fs, repetition frequency of 76 MHz, with the power fixed at 500 mW. The fluorescence spectrum is measured by fluorescence spectrometer and the fluorescence decay curves are recorded by single photon counter. Besides, the steady state photoluminescence is also studied with a JASCO FP-6500 Fluorescence Spectrometer. The result shows that with the increase of spectral overlap of the CdTe emission spectrum and the Rhodamine B absorption spectrum, the FRET efficiency of the QDs-RhB system becomes higher. Specifically, the fluorescence intensity of QDs decreases and the lifetime of QDs becomes shorter while RhB shows the opposite tendency. By means of the Förster theory of energy transfer, the spectral overlap integral J(λ), Foster radius R0 and the FRET efficiency E are calculated and the FRET characteristics of QD-RhB system is characterized. Theoretical analysis reveals that the physical source is the increase of the sample’s Forster radius. Moreover, the relationship between the ratio of acceptor/donor concentration and the FRET efficiency is investigated experimentally. When the ratio of acceptor/donor concentration increases, the lifetime of QDs turns shorter, and the FRET efficiency of the QDs-RhB system becomes higher. The two-photon excited FRET efficiency can reach 40.1% when the concentration of RhB is 3.0×10-5 mol·L-1. This study shows a brighter future in biological and optoelectronic applications.