Titanium alloys produced by additive manufacturing show excellent mechanical properties at room temperature. However, their deformation behavior at low temperature is still not fully understood. In this study, the microstructural evolution and tensile behavior of the near-α titanium alloy Ti-6.5Al-2Zr-Mo-V (TA15), fabricated via laser powder bed fusion (LPBF), were determined at both 25 °C and −196 °C. The LPBFed TA15 alloy shows the microstructure of α laths and acicular α′ martensite due to the rapid cooling rate during LPBF processing. Pyramidal <c+a> slip and numerous twins, including nano-twins and triple twins, were activated at cryogenic temperature, leading to the high ultimate tensile strength (UTS) up to 1750±8 MPa and yield strength (YS) up to 1548±25 MPa, which is higher than the strength at room temperature (YS∼1103 MPa, UTS∼1281 MPa). The dominant deformation mechanism changes from dislocation slip to twinning with decreasing temperature, leading to uniform deformation with an elongation of 5.2±0.1 %. The results can guide the control of the microstructure of LPBFed near-α titanium alloys at cryogenic temperature.