[Background] In recent years, maize stalk rot occurs widely and seriously in most corn-growing areas in China. Fusarium stalk rot not only causes an important economic loss of corn, but also is a serious threat to human and animal health because of the toxins produced by Fusarium species. [Objective] Among the complicated pathogenic fungi of corn stalk rot, Fusarium graminearum is one of the major pathogens. The infection mechanism of F. graminearum still remains an urgent research topic. [Methods] The plasmid pCAMBIA1300 was taken as the backbone, and the plasmid pCAMBIA1300-CFP-Kan, expressing the cyan fluorescent protein, was constructed using the overlapping PCR. Then the CFP gene was transformed to the genome of F. graminearum by Agrobacterium-mediated genetic transformation technology. [Results] After PCR identification and fluorescence microscopy, 31 strains labeled with CFP were acquired. [Conclusion] The pathogenicity test results suggested that F. graminearum successfully colonized in corn tissue under laser confocal observation. These results lay a solid foundation for study of the colonization of Fusarium species.