Clarifying the spatial and temporal change characteristics of regional population is helpful for promoting high-quality population agglomeration. Based on the multi-source data and by using the population gravity center method, standard deviation ellipse, population structure index, population-land distribution consistency coefficient, spatial autocorrelation analysis, and GIS spatial statistical analysis, this study analyzed the spatial-temporal characteristics of population distribution in the Lanzhou-Xining (Lan-Xi) urban agglomeration at different scales (urban agglomeration, county, township (town), and grid). The results show that: (1) The scale of population of the Lan-Xi urban agglomeration is small, population mobility is limited, urbanization level is very low, and the growth rates of population and urbanization is very slow. (2) During the study period of 2000-2017, the population gravity center moved westward slightly, and the population showed a northwest-southeast distribution pattern and formatted a core-periphery spatial structure. Lanzhou, Xining, and Linxia are the three population concentration centers, and Lanzhou has the “siphon effect” of population concentration. The population distribution is higher in the central and southern parts and lower in the northern part of Gansu Province, while in Qinghai Province the population distribution decreases outward concentrically from Xining. (3) Altitude is the main topographic factor which influences the vertical distribution pattern of population. The population is mainly concentrated in the valley basins with an altitude of less than 2400 m, indicating that the vertical gradient effect and basin agglomeration are obvious. The spatial-temporal characteristics of population distribution were analyzed from different perspectives and at different scales, so that the research results and corresponding countermeasures and recommendations can provide a scientific basis for promoting the spatial layout optimization of population distribution.