In this paper, the fatigue performance of rib-to-deck joints in orthotropic steel decks (OSDs) using thickened edge U-ribs (TEUs) and the OSD using conventional U-ribs (CUs) are investigated based on the fatigue test result and in-situ monitoring data. Firstly, comparative fatigue tests were carried out with full-scale rib-to-deck specimens. Probability–stress–life (P–S–N) curves were derived through the test data measured from a total of 18 specimens, including 7 CU specimens and 11 TEU specimens. According to the results, the TEU can lead to a notable enhancement in the fatigue strength of rib-to-deck joints, i.e., 21.4% in terms of nominal stress and 21.1% in terms of hot spot stress. After that, a typical OSD steel bridge was selected as the prototype to investigate the fatigue performance of rib-to-decks in the OSD using TEUs under actual applications. In the analysis, the uncertainty in both fatigue strength, and vehicle loads were considered. A multi-scale finite element model of the prototype bridge was established, and numerical analysis conducted to derive the vehicle-induced stress spectra of the rib-to-deck joints in critical positions. In the derivation, a stochastic traffic model was employed, through which the in-situ measurement was incorporated. Finally, fatigue reliability analysis was carried out for the prototype bridge based on the above works. The result showed that a notably higher fatigue reliability can be expected in the rib-to-deck joint in OSDs using TEUs when compared with the joints in OSDs using CUs, which in turn can lead to notable improvement in fatigue life. For instance, under the target reliability of 2.3, the fatigue life of the two critical rib-to-deck joints were, respectively, increased by 153% and 155% when using TEUs.