The present paper is focused on studying the effect of heat input on the tensile damage evolution of pulsed Nd:YAG laser welding of Ti6Al4V alloy under monotonic loading. To analyze the reasons that the tensile fracture site of the pulsed-laser-welded Ti6Al4V sheet joints changes with the heat input under monotonic loading, the microstructure of the sample with different nominal strain values was investigated by in situ observation. Experiment results show that the tensile ductility and fatigue life of welded joints with low heat input are higher than that of welded joints with high heat input. Under tensile loads, the critical engineering strain for crack initiation is much lower in the welded joint with high heat input than in the welded joints with low and medium heat input. And the microstructural damage accumulation is much faster in the fusion zone than in the base metal for the welded joints with high input, whereas the microstructural damage accumulation is much faster in the base metal than in the fusion zone for the welded joints with low input. Consequently, the welded joints fractured in the fusion zone for the welds with high heat input, whereas the welded joints ruptured in the base metal for the welds with low heat input. It is proved that the fine grain microstructure produced by low heat input can improve the critical nominal strain for crack initiation and the resistance ability of microstructural damage.