Plant responses to drought stress are complex due to various mechanisms of drought avoidance and tolerance to maintain growth. Traditional plant phenotyping methods are labor-intensive, time-consuming, and subjective. Plant phenotyping by integrating kinetic chlorophyll fluorescence with multicolor fluorescence imaging can acquire plant morphological, physiological, and pathological traits related to photosynthesis as well as its secondary metabolites, which will provide a new means to promote the progress of breeding for drought tolerant accessions and gain economic benefit for global agriculture production. Combination of kinetic chlorophyll fluorescence and multicolor fluorescence imaging proved to be efficient for the early detection of drought stress responses in the Arabidopsis ecotype Col-0 and one of its most affected mutants called reduced hyperosmolality-induced [Ca2+]i increase 1. Kinetic chlorophyll fluorescence curves were useful for understanding the drought tolerance mechanism of Arabidopsis. Conventional fluorescence parameters provided qualitative information related to drought stress responses in different genotypes, and the corresponding images showed spatial heterogeneities of drought stress responses within the leaf and the canopy levels. Fluorescence parameters selected by sequential forward selection presented high correlations with physiological traits but not morphological traits. The optimal fluorescence traits combined with the support vector machine resulted in good classification accuracies of 93.3 and 99.1% for classifying the control plants from the drought-stressed ones with 3 and 7 days treatments, respectively. The results demonstrated that the combination of kinetic chlorophyll fluorescence and multicolor fluorescence imaging with the machine learning technique was capable of providing comprehensive information of drought stress effects on the photosynthesis and the secondary metabolisms. It is a promising phenotyping technique that allows early detection of plant drought stress.