The temperature fluctuation in a single-phase microchannel heat sink (MCHS) is investigated using the integrated temperature sensors with deionized water as the coolant. Results show that the temperature fluctuation in single phase is not negligible. The causes of the temperature fluctuation are revealed based on both simulation and experiment. It is found that the inlet temperature fluctuation and the gas bubbles separated out from coolant are the main causes. The effect of the inlet temperature fluctuation is global, where the temperatures at different locations change simultaneously. Meanwhile, the gas bubble effect is localized where the temperature changes at different locations are not synchronized. In addition, the relation between temperature fluctuation and temperature gradient is established. The temperature fluctuation increases with the temperature gradient accordingly.