Abstract Centrifuge rotors are designed for high service life duty and should stand against 2 times the maximum operation speed in a spin tester in terms of the manufacture’s standard. A centrifuge rotor prototype had burst before reaching 2 times the maximum operation speed in an over-speed spin testing, therefore, failure analysis was required to determine the cause of the burst and recommendations for the structure optimization were needed to improve the rotor integrated strength. After the chemical analysis and the microstructure identity, a finite element model of 1/24 rotor under centrifugal load was adopted for mechanical stress analysis. The analysis indicated that local stress peaks occurred on the top round between the receptacle and the hub and on the edges of the tube cavities. By comparing computational and experimental results, it is revealed that the rotor fractured on two regions one after another when the peak stress exceeded the ultimate strength of the material. Consequently, some recommendations were made for the structure optimization to improve the rotor safety performance. The peak stress was reduced below the allowable material strength margin with increasing the fillet radius of the round and decreasing the depth of the liquid gather groove. The burst of the redesigned rotor was eliminated in the later over-speed spin testing.