For the first time, multifunctional magnetic particles based rare cell isolation is demonstrated combined with the downstream laser desorption/ionization mass spectrometry (LDI-MS) to measure the metabolism of enriched circulating tumor cells (CTCs). The characterization of CTCs metabolism plays a significant role in understanding the tumor microenvironment, through exploring the diverse cellular process. However, characterizing cell metabolism is still challenging due to the low detection sensitivity, high sample complexity, and tedious preparation procedures, particularly for rare cells analysis in clinical study. Here, ferric oxide magnetic particles are conjugated with anti-epithelial cell adhesion molecule on the surface for specific, efficient enrichment of CTCs from phosphate buffered saline and whole blood with cells concentration of 6-100 cells per mL. Moreover, these hydrophilic particles as matrix enable sensitive and selective LDI-MS detection of small metabolites (M-w < 500 Da) in complex biomixtures and can be further coupled with isotopic quantification to monitor selected molecules metabolism of approximate to 50 CTCs. This unique approach couples the immunomagnetic separation of CTCs and LDI-MS based metabolic analysis, which represents a key step forward for downstream metabolites analysis of rare cells to investigate the biological features of CTCs and their cellular responses in both pathological and physiological phenomena.