Chlorophyll fluorescence kinetics is closely related to the algae photosynthesis and is a natural tool to detect viable algae cells. Compared with the common method of combining fluorescein staining in ballast water with the microscopic examination of viable algae cells number, chlorophyll fluorescence kinetics has the characteristics of rapid measurement, high sensitivity, and no need for pretreatment. However, there are a large number of dead algae cells and colored dissolved organic matter (CDOM) in marine ballast water, and the fluorescence background is complex. Therefore, obtaining the photosynthetic fluorescence parameters that are not affected by the fluorescence background and can accurately characterize the viable algae cells number is the key to directly measuring the viable algae cells number in ballast water through chlorophyll fluorescence kinetics. In this paper, with the dead cells and CDOM solution simulating the complex fluorescence background of ballast water and the 3-(3c,4-(dichlorophenyl)-1,1-dimethylurea (DCMU) stress condition simulating the practical application, the relationship of various photosynthetic fluorescence parameters such as F_m, F₀, [RCII], and F_v with the viable algae cells number was investigated at different fluorescence backgrounds. The results show that F_m, F₀, and [RCII] are all affected by the dead cells number and the CDOM concentration to varying degrees, and only F_v is not affected by the fluorescence background, with a relative standard deviation being less than 5%. In an experiment of diluting the solution of viable algae cells with different diluents, F_v and the viable cells number have a good linear correlation, with the correlation coefficient R² above 0.98. Under the stress of DCMU, only F_v displays an excellent positive correlation with the viable algae cells number, and the correlation coefficient is 0.986. These results demonstrate that F_v is not disturbed by the fluorescence background and is the best photosynthetic fluorescence parameter to characterize the number of viable algae cells in ballast water.