Separate impacts of building and tree on the urban thermal environment have been studied extensively, but their combined impacts, especially from both the horizontal (i.e., two-dimensional (2D)) and vertical (i.e., three-dimensional (3D)) perspectives remain largely unclear. Based on satellite thermal data and elaborate 2D and 3D urban morphology, herein we simultaneously investigate the separate and combined impacts of building and tree over Nanjing in China from both the 2D and 3D perspectives. We further examine the day–night contrast together with the sensitivity of such impacts to scale. Our results show that, when compared with urban structures from a single dimension, the combination of 2D and 3D structures is more capable of predicting urban land surface temperatures (LSTs) for both day and night. The assessments further illustrate that the separate and combined impacts of building and tree on LSTs are usually more significant when the spatial scale increases. As for the separate impacts of building and tree, 2D structure affects more urban thermal environment than 3D structure at all spatial scales during the day, but an opposite trend occurs at night. Moreover, for the combined impact of building and tree on LST across different scales, daytime and nighttime LSTs are respectively dominated by 2D and 3D building structures. Combining 2D and 3D structures improves the explained LST variation by 7.3%–11.1% and 25.3%–37.7% for day and night, respectively when compared to using 2D structures only. These findings emphasize the need to incorporate both 2D and 3D urban morphology to improve the urban thermal environment.