Phosphorylation of eIF2α is an important strategy for living organisms to adapt to metabolic and physiological changes that are often associated with external stimuli. GCN2 is one of the well-studied eIF2α kinases in yeast and mammals, which is responsible for the survival of the organism under amino acid starvation. Despite the downstream reactions being quite divergent, AtGCN2 exhibits a high primary sequence similarity to its yeast and animal counterparts. In this study, we provide experimental evidence to show that AtGCN2 shares similar biochemical properties to the yeast and animal homologues. Our in vitro assays demonstrate the binding of the C-terminus of AtGCN2 to uncharged tRNA molecules and the enzymatic activities of AtGCN2 on both eIF2α homologues in A. thaliana, thus providing essential information for further understanding the functions of plant general control non-repressible (GCN) homologues.