Autotrophic nitrogen fixation genes and an autotrophic nitrogenase expression cassette for plants and uses thereof are disclosed. Nitrogenase related genes of nitrogen fixation gene cluster from Paenibacillus sp. are specifically optimized by plant expression patterns through synthetic biology, to obtain autotrophic nitrogen fixation genes, which are then constructed into plant constitutive expression units. The expression units are assembled in a plant expression vector by isocaudarner cloning method and transformed into plants, thereby realizing autotrophic nitrogen fixation of the plants.