Defects on external surface of in-service hydraulic tubes can reduce total life cycles for operation. Evaluation of fatigue life of the tubes with damage is thus critical for safety reasons. A methodology of generating defects in the Ti-3Al-2.5V tube—a widely used pipeline in hydraulic systems of aircrafts—using nanosecond pulsed laser for experimental fatigue life study is described in this paper. Straight tubes of five different sizes were laser micromachined to generate notches of given length and depths on the outside surface. Approaches were developed to precisely control the notch dimensions. The laser-notched tubes were tested with cyclic internal impulse pressure and fatigue life was measured. The laser notches and fatigue cracks were characterized after the test. It is concluded that laser micromachining generated consistent notches, and the influence of notch depth on fatigue life of the tube is significant. Based on the experimental test results, the relationship between the fatigue life of the Ti-3Al-2.5V tube and the notch depth was revealed. The research demonstrated that laser micromachining is applicable for experimental fatigue life study of titanium tubes. The presented test data are useful for estimating the damage limits of the titanium tubes in service environment and for further theoretical studies.