Plant breeding is important for increasing the yield and improving the quality of crop. Modern techniques of plant breeding require various phenotypic traits obtained from a large number of crops in order to cultivate varieties with useful traits. Researchers have been interested in developing nondestructive, fast and efficient plant phenotyping techniques, which could bridge the knowledge gap between the plant phenotype and the genotype. The chlorophyll fluorescence imaging can provide information related to the plant photosynthesis as one of the useful tools in the current phenotyping techniques, which can be used in plant responses to biotic and abiotic stresses and promote the selection of useful plant traits. This paper presents a brief introduction of fluorescence imaging system, methods of fluorescence image analysis and applications in plant phenotyping, and demonstrates challenge issues, technical solutions and potential applications of chlorophyll fluorescence imaging in plant phenotyping.