Titanium alloy is a high-performance material widely used in aerospace, nuclear engineering, petrochemical equipment, and other fields. In order to overcome the shortcomings of the traditional uniaxial tensile test to test the anisotropic mechanical properties of titanium alloys that require large-size specimens, this paper proposes to use the single-stress small punch bar test method to study the anisotropic tensile properties of rolled Ti-6Al-4V titanium alloys based on the test method of micro-specimens. In order to verify the accuracy of the single-stress small punch bar test method, standard uniaxial tensile tests as well as standard small punch bar tests were also carried out, and the results of the standard uniaxial tensile tests showed that the rolled Ti-6Al-4V titanium alloys tested in the experiments had significant anisotropy. Comparison of the tensile test with the standard small punch bar test reveals that the standard small punch bar test cannot accurately test the anisotropic mechanical properties of titanium alloys due to the effect of bi-directional stresses. In addition, the single-stress small punch bar test method can test the anisotropy of the rolled Ti-6Al-4V titanium alloy used in the test with high accuracy. The average deviation of the two tests for the yield strength in direction Ⅰ (perpendicular rolling direction) is 2.14%, that of the two tests for the direction Ⅱ (rolling direction) is 3.57%, and that of the two tests for the direction Ⅲ (thickness direction) is 2.14%; the average deviation of the two tests of tensile strength in direction Ⅰ is 3.60%, the average deviation of the two tests in direction Ⅱ is 3.53%, and the average deviation of the two tests in direction Ⅲ is 5.34%. The small deviations of the test results in each direction measured by the two test methods indicate that the use of the single-stress small punch bar test method to test the tensile properties of Ti-6Al-4V titanium alloy in different directions has high accuracy.