Strong and rapid responses of soil microbial respiration to pulses, such as those from available soil organic matter (SOM) or water input from precipitation (especially in arid areas), are common. However, how soil microbes utilize new SOM inputs and the effects that temperature may have on their activities are unclear owing to the limitation in the application of traditional isotopic techniques at minute scales. In the present study, we developed a system of measuring ¹²CO₂ and δ¹³C minutely and synchronously under controlled incubation temperatures, i.e., for 48 h at 7, 10, 15, 20, and 25 °C, to explore the carbon utilization strategies of soil microbes. We measured the respiration rates of soil microbes in response to different carbon sources, i.e., added glucose (Rg) and initial SOM (Rs), as well as the total respiration rate (Rt). All responses were rapid and characterized by unimodal curves. Furthermore, the characteristic values of these curves, such as the maximum of rate (R-max), the time required to achieve R-max, and the ratio of the duration of R-max to that of 1/2 R-max, were all dependent on incubation temperature. Interestingly, temperature greatly influenced the strategy that microorganisms employed to utilize different carbon sources. The effects of temperature on the intensity of the microbial respiratory response and the ratio of Rg/Rs are important for evaluating the effect of land-use changes or variations in seasonal temperature on SOM turnover and should be considered in ecological models in future studies.