In situ microstructural observations on slip lines and microfracture processes have been coupled with mechanical deformation to investigate the acoustic emission of Ti-6Al-4V, a two-phase alloy. The material has been subjected to several different heat treatments to produce various microstructures and grain sizes. The dependencies of acoustic emission (AE) characteristics, such as the AE events amplitude, the rate of AE, location of the AE sources and the total events of AE in each deformation test, on work hardening, grain size, second phase content and morphology, and strain rate have been explored. The presence of the second phase, beta, existing in either an intergranular network or thin plates, was found to have little influence on the AE of this material. Furthermore, it was found that the dependencies of the AE characteristics on work hardening, grain size, second phase and strain rate could be explained in terms of the operation of Frank-Read dislocation sources.