Abstract: In order to solve the problem of sample fixation in two photon intravital imaging, a set of small animal fixation device suitable for a variety of different modes has been developed. The device can be flexibly adjusted according to the size of the animal, and can suitable for fixing different regions of interest (such as brain, abdomen, limbs, etc.) of small animals to ensure the stability of the imaging, and located the imaging position for long time and multiple nodes imaging. The device is low-cost, easily productive and broadly suitable, which provides a multifunctional immobilization method of intravital imaging for small animals. The solution will extremely help researchers to promote the quality of most intravital experiments on small animals under anesthesia status.