This paper reports the demonstration of efficient single molecule detection in flow cytometry by two-photon fluorescence excitation. We have used two-photon excitation (TPE) to detect single DNA fragments as small as 383 base pairs (bp) labeled with the intercalating dye, POPO-1, at a dye:nucleotide ratio of 1:5. TPE of the dye-DNA complexes was accomplished using a mode-locked, 120 fs pulse width Ti:sapphire laser operating at 810 nm. POPO-1 labeled DNA fragments of 1.1 kilobase pairs (kbp) and larger were sequentially detected in our flow cytometry system with a detection efficiency of nearly 100%. The detection efficiency for the 383 bp DNA fragments was approximately 75%. We also demonstrate the ability to distinguish between different sized DNA fragments in a mixture by their individual fluorescence burst sizes by TPE. These studies indicate that using TPE for single molecule flow cytometry experiments lowers the intensity of the background radiation by approximately an order of magnitude compared to one-photon excitation, due to the large separation between the excitation and emission wavelengths in TPE.