A new biphenyl ethylene derivative, 4,4′-bis-(2-(4-N-ethyl-N-ethoxyl-amino) styryl] biphenyl (BEEASP) was successfully synthesized and studied as a potential fluorescent probe of DNA detection. The linear absorption and fluorescent spectra of the dye in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye–DNA complex were also examined to evaluate their photophysical properties. The binding constant of dye–DNA was calculated with a moderate value. Two-photon active cross-sections (Фδ) were finished by the reference method, and the value at a stable complex of dye–DNA was about 37GM. The experimental results showed that BEEASP can be used as an efficient fluorescent probe to detect DNA by single-photon excitation and two-photon excitation. The fluorescent method date indicated that the mechanism of dye–DNA complex interaction may be the groove binding.