Multidirectional isothermal forging (MIF) experiments of Ti₂AlNb-based alloy was carried at 800°C. The microstructure evolution and hardness of the alloy under different deformation cycles were quantitatively analyzed by SEM, XRD, EBSD and hardness testing. The dynamic spheroidization behavior of the lath O and alloy hardening mechanism was revealed. The results show that as the number of deformation cycles increases, the initial coarse lath O has undergone two refinement mechanisms, namely dynamic mechanical breaking and dynamic recrystallization. The globalization of the lath mainly occurs in 3 cycles deformation process. The dynamic spheroidization process is accompanied by four deformation behaviors: bending, kinking, shearing and grain tearing. The mechanism of the latter two types of deformation can be summarized as: O/O phase boundary separation、penetration of B2 phase、O phase separation and O phase spheroidization. The initial lath O phase is refined to 1.3 μm equiaxed structure, and the percentages of fine equiaxed O phase is as high as 68.84% after 3 cycles of MIF. At this time, the hardness of the alloy is 103.55HV higher than the initial sample, and the contribution of grain boundary hardening mechanism is 64%.