Objective：This study aims to introduce the manufacture and use instruction for a simple and multifunctional device for mouse fixation. Methods：Discarded 50 mL centrifuge tubes were collected and processed into the fixation device according to the pre-designed program. A total of 60 mice were randomly and equally assigned into two groups and fixed with traditional methods（control group）and this fixation device（study group），respectively. Tail vein injections or tail-clamping blood sampling or intra-articular injections were performed. The procedure durations and success rate were recorded. Results：A single 50 ml centrifuge tube weighed about 12 g and mere 15 minutes were taken to manufact the fixation device. The procedure durations of tail vein injection，tail-clamping blood sampling and intra-articular injections in control and study groups were（32.10±1.97）s and（30.27±2.20）s，（30.10±1.73）s and（28.67±1.55）s，and（40.77±2.73）s and（38.73±2.16）s，respectively. The data between two groups were significantly different（P＜0.01）. The success rates of the three procedures in control group study group were 100% and 100 %，100% and 100%，and 83.33% and 95.00%，respectively. There were significant differences in procedure duration and success rate between two groups（P＜0.05）. Conclusion：The mouse fixation device possesses the advantages of easily available materials，simple manufacture，convenient transportation and multiple functions. It provides a new safe and reliable choice for mouse fixation in the experiment and is worthy to be promoted.