(1) Background: As the principal structural element of an unmanned aerial vehicle (UAV), a lightweight, high-performance wing-fuselage connection structure plays an important role in UAV structural integrity. Previous studies focused on the static strength characteristics of aluminum and high-strength steel wing-fuselage connection structures. With the widespread usage of titanium material in aviation, research should address the fatigue performance of titanium wing-fuselage connection structures. This paper aims to investigate the fatigue performance of the titanium wing-fuselage connection structure by using analysis and experimental methods, and to develop a reliable fatigue assessment method based on the experimental results. (2) Methods: General and detail finite element models were established, and the stress distribution at the detail fatigue design point was obtained via finite element analysis. Based on the equivalent life curve theory, a dimensionless value, detail fatigue value (DF), was proposed to characterize the fatigue performance for structure. By using detail fatigue value (DF) method, the theoretical fatigue performance value was calculated. The fatigue test of the wing-fuselage connection structure was conducted, and the fatigue life was determined according to the test results. (3) Results: For the wing joint with fatigue failure in the test, the test fatigue performance was close to the theoretical value. It can be concluded from the DF value that the analysis and test results are consistent, and the values obtained from the analysis are more conservative than test results. (4) Conclusions: The error between the test DF value and the theoretical analysis DF value is small, and the theoretical analysis of fatigue performance can represent the fatigue characteristics of the wing-fuselage connection structure. The detail fatigue value (DF) method can predict the fatigue performance of the structure quite well.