Abstract: Free fatty acids (FFAs) play a vital role in human physiological activities, and as tumor detection markers, FFAs have significant advantages in sensitivity and specificity. It is of great significance for accurate determination of FFAs in serum for scientific research and clinical diagnosis. This study developes a new detection method of rapid, highly selective and high-throughput analysis for FFAs in serum based on the advantages and limitations of matrix assisted laser desorption-mass spectrometer (MALDI-MS) and stimuli responsive molecularly imprinted membrane (Sr-MIM) technology. The technique provides a new idea for the trace analysis of small molecular compounds in complex samples by MALDI-MS. At the same time, it broadens application scope of large instruments, which is conducive to improving the sharing efficiency of advantageous resources. It has been deeply applied in college students’ innovation training programs to stimulate students’ academic interest and innovation potential, improve the quality of training top-notch innovative talents, and create a free and innovative scientific research ecological environment.