The uneven temperature distribution will cause significant thermal stress and deformation of concrete box girder, which will further endanger the structural safety. Based on the monitoring data of temperature and stress of a concrete box girder bridge in service, the temperature distribution characteristics and stress evolution law of structure are amply analyzed in this paper. The results indicate that there are obvious uneven temperature distribution in the vertical direction of the box girder structure and along the thickness of the roof and web. Simultaneously, the temperature and strain of the box girder structure have obvious periodic changes on the annual and daily time scales. Subsequently, the correlation between temperature and strain is preliminarily investigated, it is found that the Spearman correlation coefficient reached 0.91. On basis of this, the monitoring strain data are separated into different frequency components by wavelet transform, in which layer a6 and layer d6 were significantly correlated with seasonal and daily temperature variations, respectively. Therefore, the effect mechanism of temperature on strain is further revealed. In general, the research results are of guiding significance to enhance the safety and durability of bridge structures during operation.