In order to evaluate the fatigue life of a steel bridge deck, stress measuring points were set near the weld of the roof connected to the U-rib and the arc-shaped notch of the diaphragm plate across a quarter of a bridge’s steel box girder, and the fatigue stress was monitored for 24 hours. According to the measured data, the fatigue stress spectra were calculated with the rain-flow counting method. Based on the BS 5400 specification, the fatigue damage state of the stress measuring points was evaluated. The results show that the stress level of the measuring points near the arc-shaped notch of the diaphragm plate was relatively high. Each stress measuring point was mainly a low-rising stress cycle and the cycles of 2 to 10 Mpa stress amplitude accounted for more than 90%. However, the maximum stress amplitude exceeded the limit of fatigue damage stipulated by the BS 5400 specification. Fatigue life analysis shows that fatigue cracks in the weld of the roof connected to the U-rib easily occurred near the roof area at the welding hole, and the fatigue life of the weld of the diaphragm plate connected to the U-rib’s web was short.