This study investigates the stress relaxation behavior and associated microstructure evolution in laser melted deposited Ti–6Al–4V alloy under various temperature and pre-strain conditions for the first time. A set of stress relaxation and tensile tests has been performed to characterize the stress and strength evolution properties of the as-deposited alloy, and microstructural analysis has also carried out for mechanisms identification. It is found that the as-deposited alloy exhibits a lower threshold stress (less than 10 MPa), faster relaxation rate, and a more significant decrease in strength (9.9%∼15.8%) when compared with the same testing conditions of conventionally extruded alloy. Based on the microstructural evolutions, as well as the stress exponent analysis, the stress relaxation mechanisms have been identified: at low pre-strain and temperature (700 °C and 0.5%), diffusion creep plays the dominant role, while with the increasing pre-strains and temperatures, the mechanism transits to dislocation climb, and eventually to grain boundary sliding, at 750 °C and 10%. It is observed that the fine lamellae in the as-deposited alloy are susceptible to morphological changes during relaxation, including dislocation rearrangement, lamellae spheroidization and coarsening. These microstructural evolutions lead to the creep mechanism transitions, as well as the noticeable reduction in yield strength after relaxation. The results could provide insights into designing appropriate stress relaxation parameters for deposited alloys in hybrid additive manufacturing processes.