It is well-known that grain refiners can tailor the microstructure and enhance the mechanical properties of titanium alloys fabricated by additive manufacturing (AM). However, the intrinsic mechanisms of Ni addition on AM-built Ti–6Al–4V alloy is not well established. This limits its industrial applications. This work systematically investigated the influence of Ni additive on Ti–6Al–4V alloy fabricated by laser aided additive manufacturing (LAAM). The results showed that Ni addition yields three key effects on the microstructural evolution of LAAM-built Ti–6Al–4V alloy. (a) Ni additive remarkably refines the prior-β grains, which is due to the widened solidification range. As the Ni addition increased from 0 to 2.5 wt. %, the major-axis length and aspect ratio of the prior-β grains reduced from over 1500 μm and 7 to 97.7 μm and 1.46, respectively. (b) Ni additive can discernibly induce the formation of globular α phase, which is attributed to the enhanced concentration gradient between the β and α phases. This is the driving force of globularization according to the termination mass transfer theory. The aspect ratio of the α laths decreased from 4.14 to 2.79 as the Ni addition increased from 0 to 2.5 wt. %. (c) Ni as a well-known β-stabilizer and it can remarkably increase the volume fraction of β phase. Room-temperature tensile results demonstrated an increase in mechanical strength and an almost linearly decreasing elongation with increasing Ni addition. A modified mathematical model was used to quantitatively analyze the strengthening mechanism. It was evident from the results that the α lath phase and the solid solutes contribute the most to the overall yield strength of the LAAM-built Ti–6Al–4V–xNi alloys in this work. Furthermore, the decrease in elongation with increasing Ni addition is due to the deterioration in deformability of the β phase caused by a large amount of solid-solution Ni atoms. These findings can accelerate the development of additively manufactured titanium alloys.