Understanding the spatial variation law of soil respiration (Rs) and its influencing factors is very important when simulating and predicting the terrestrial carbon cycle process. However, there are still limitations in understanding how different sampling scales affect the spatial heterogeneity of Rs and whether the spatial scale effect will change with habitat types. Our objectives were to explore the effects of different sampling scales on the spatial variability of Rs and the relative importance of soil abiotic characteristics and plant traits in influencing the spatial variability of Rs. The Rs, soil properties, and plant traits were measured through field investigation and indoor analysis in the Tugai forest desert plant community in the Ebinur Lake Basin in northwest China. The Rs showed significant water gradient changes, with a coefficient of variation of 35.4%–58%. Plot types had significant effects on Rs, while the change of sampling scale did not lead to significant differences in Rs. At the plot scale, Rs spatial variation at the 5 m × 5 m sampling scale mainly depended on plant traits (leaf length, leaf thickness, leaf dry matter content, and leaf phosphorus content, p < 0.05), while Rs spatial variation at the 10 m × 10 m scale mainly depended on soil properties (soil total phosphorus, ammonium nitrogen, soil water content, and pH, p < 0.05). At the local scale, soil nutrients (soil available phosphorus and ammonium nitrogen) and plant traits (maximum plant height, leaf length, and phosphorus content) at the 5 m × 5 m scale jointly explained 49% of the spatial change of Rs. In contrast, soil microclimate (soil water content), soil nutrients (soil pH, available phosphorus, and nitrate nitrogen), and plant traits (leaf thickness) jointly explained 51% of the spatial variation of Rs at the 10 m × 10 m scale. These results demonstrate the potential to predict the spatial variability of Rs based on the combination of easily measured aboveground functional traits and soil properties, which provides new ideas and perspectives for further understanding the mechanism of Rs change in Tugai forests.