Abstract: Schizosaccharomyces pombe is an excellent model organism for studying mitochondrial mechanism. To establish a suitable fluorescent tag indicating the mitochondrial morphology for S. pombe, the plasmid system pMTS7 which was boned on plasmid pYJ19 with a nmt41 promoter, EGFP as reporter, the mts derived from S. pombe’s own protein Cox4 as a mitochondrial targeting sequences and leu1 as selection marker, respectively,was constructed.pMTS7 was transformed into S. pombe named y HL6381, and MitoTracker was used as a control. The results of fluorescence microscopy showed that pMTS7 could be co-located with MitoTracker and correctly display mitochondrial localization. Compared with the MitoTracker which has some toxic and is difficult to control, using pMTS7 to mark mitochondria can observe clear mitochondrial morphology under a microscope for a long time, and can see the dynamic changes of mitochondrial fission and fusion. The successful construction of pMTS7 provided a technical tool for studying mutants of mitochondrial genes in S. pombe.