Narrow gap laser welding (NGLW) is a common solution for the welding of thick structures. NGLW was carried out on narrow-gap butt joints of 40 mm-thick Ti-6Al-4V alloy plates with a U-shaped groove. The distribution characteristics of the interlaminar microstructure in different height ranges of the joint were investigated, and the evolution behavior and formation mechanism of the interlaminar microstructure of the joint were also revealed. This showed that a large amount of short needle martensite nucleated and grew up near the fusion line and the upper boundary of the remelting zone. The "softening" phenomenon occurred in all welds except the cover layer weld. The microstructure evolution and defect migration, induced by multiple welding thermal cycles in the upper weld forming process, were the main reasons for the "softening" of the lower weld. The tensile strength of each sample changed in the range of 920~990 MPa; the fracture mode of the sample belongs to a transgranular ductile fracture. In addition, compared with the upper part of the joint, the plasticity and toughness of the weld area in the lower part of the joint was improved.