Abstract: β-stable element segregation defects (β fleck) significantly impact on the mechanical properties of near-β-type titanium alloys. In order to reveal the influence of β flecks on the mechanical properties of TB6 titanium alloy, TB6 titanium alloys with and without β flecks were subjected to mechanical testing at room temperature. In situ tensile tests, scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) were used to analyze the microstructure and the evolution of microstructure to investigate the mechanism of β flecks’ influence on mechanical properties. The specimens with β flecks had higher tensile strength and yield strength, 1 170 MPa and 1 107 MPa, respectively, showing an increase of 3.90% and 3.79% compared to those without β flecks defects. The elongation and cross-sectional shrinkage of the specimens with β flecks were 5.20% and 15.00%, respectively, reduced by 66.67% and 22.58% compared to those without β flecks. Due to the enrichment of Fe element in the TB6 titanium alloy, the content of equiaxed α phase in the β fleck area was relatively low and the content of lamellar α phase was high, leading to the higher tensile strength for TB6 titanium alloy with β flecks. The in-situ notch tensile test showed that apparent cracks were observed in the samples without β flecks when the loading force reached 1 937 N, and obvious cracks were observed in the samples containing β flecks when the loading force reached 1 760 N. As the force increased, the cracks further expanded until fractured, indicating that β flecks significantly affected on the strength and ductility of TB6 titanium alloy.