Hot deformation behaviors and microstructure evolution of Ti-3Al-5Mo-4Cr-2Zr-1Fe (Ti-35421) alloy in the β single field are investigated by isothermal compression tests on a Gleeble-3500 simulator at temperatures of 820-900 °C and strain rates of 0.001-1 s^-1. The research results show that discontinuous yield phenomenon and rheological softening are affected by the strain rates and deformation temperatures. The critical conditions for dynamic recrystallization and kinetic model of Ti-35421 alloy are determined, and the Arrhenius constitutive model is constructed. The rheological behaviors of Ti-35421 alloys above β phase transformation temperature are predicted by the constitutive model accurately. The EBSD analysis proves that the deformation softening is controlled by dynamic recovery and dynamic recrystallization. In addition, continuous dynamic recrystallization is determined during hot deformation, and the calculation model for recrystallization grain sizes is established. Good linear dependency between the experimental and simulated values of recrystallized grain sizes indicates that the present model can be used for the prediction of recrystallized grain size with high accuracy.