The temperature sensitivity (Q₁₀) of soil microbial respiration (R_s) is a critical parameter for predicting soil carbon (C) fluxes under changing climatic conditions, and labile C is continuously input into soil via root exudates or plant litter in field. However, how Q₁₀ responds to labile C input remains uncertain, especially across large geographical regions. We collected eight soils from tropical to cold-temperate forests along a north–south transect in eastern China, and soils were added with either water as a control or glucose as an analogue for labile C input to investigate how Q₁₀ responds to labile C input at a large geographical scale. Then, soils were incubated under varying temperature conditions from 5 to 30 °C for 2-weeks, and R_s was measured at a high frequency (each sample was measured at 20 min intervals within 12 h) after 1 d, 7 d and 14 d incubation. R_s and Q₁₀ were then calculated for both the control and glucose addition treatments. Compared with those in the control, glucose addition significantly increased R_s and Q₁₀ in all forest soils. The increase in Q₁₀ (ΔQ₁₀) between the control and glucose addition treatments differed significantly among the eight forest soils and was the highest in mid-latitude regions. Furthermore, ΔQ₁₀ was significantly and negatively correlated with the soil C availability index (CAI), indicating that the Q₁₀ of soils with lower basal substrate availability (e.g., soils in mid-latitudes of eastern China) are enhanced more by labile C input than those of higher basal substrate availability. A significant negative relationship between Q₁₀ and substrate quality (SQI, measured as basal microbial respiration rate at 0 °C) without glucose addition supported the carbon quality temperature hypothesis; however, this hypothesis was not always valid after adding glucose. Our findings highlight the importance of basal substrate availability in influencing Q₁₀ after labile C addition, suggesting that C-climate models should incorporate different scenarios of labile C input in the future.