The interaction between dislocations and helium-induced defects in Ti-5331 alloy with different cold rolling deformations (0%, 10%, 25%), and the retention behavior of helium in the alloy were investigated by positron annihilation spectroscopy (PAS), transmission electron microscopy (TEM), and grazing incidence X-ray diffraction (GIXRD). The results show that the dislocations generated by deformation in Ti-5331 alloy hinder the diffusion of injected helium atoms into the alloy. Moreover, a large number of vacancy defects generated by irradiation in the alloy form helium vacancy complexes with helium atoms, which finally evolve into helium bubbles. The irradiation damage of the inner layer of Ti-5331 alloy after deformation is lower than that of the surface layer. Additionally, the helium desorption amounts of the alloy with deformations of 0%, 10%, and 25% are 1.576×10¹⁵, 1.894×10¹⁵, and 2.171×10¹⁵ ions/cm², respectively, indicating that the larger the deformation, the more the helium retention in the alloy.