The utilization of enzyme-induced calcium carbonate precipitation (EICP) to consolidate aeolian sand has received significant attention in recent years. However, urease activity is directly affected by temperature, which varies greatly from day to night, especially in desert areas. To investigate the effect of alternating temperature on aeolian sand cementation by EICP, three experimental groups were designed to simulate the sunrise-to-sunset cycle in a natural desert environment: T1 (a process from heating to constant temperature to cooling), T2 (a process from cooling to constant temperature to heating), and T3 (a process of constant temperature throughout) as a control group. The differences in calcium carbonate content, precipitation rate of calcium carbonate, permeability coefficients, and shear wave velocity were compared and analyzed. Meanwhile, scanning electron microscopy (SEM) was conducted to observe the external cementation states by mineralization. The results showed that T2 had the highest calcium carbonate content, followed by T3 and, finally, T1, which were also confirmed by permeability coefficient and shear wave velocity tests. In addition, different alternating temperature processes would affect the survival time of the urease, and T2 showed the longest reaction time as the urease stayed active for the longest time in this process. The results provide a scientific reference for the selection of construction periods in which EICP can be optimally applied for the on-site aeolian sand cementation.