The complex composition, size, and distribution of microstructures of titanium (Ti) alloy affect the mechanical properties of titanium alloy and its application in aerospace, ocean technology, and bioengineering. In this paper, the microstructural components and mechanical behavior of Ti80 are first investigated experimentally. According to the experimental observations of the dual-phased microstructures, a mechanism and microstructure-based constitutive model of Ti80 is established to study the quantitative relationship between mechanical behavior and equiaxed αp + lamellar αs + β microstructures of titanium alloys. And the influence of dislocation evolution and accumulation on the strengthening and work-hardening of materials is also explored in detail, especially the contribution of dislocation pile-up zone at the phase boundary between α phase and β phase on the strengthening of materials. Numerical results show that the proposed model can describe the constitutive behavior of Ti80 very well, including yield stress and strain hardening. And various strengthening mechanisms originated from the grain boundaries, phase boundaries of β transformation structure and β precipitation are analyzed. The proposed model is further applied to predict the constitutive behaviors of the titanium alloy with different sizes and various volume fractions of microstructure.