The invention provides polymorphism of a deuterium-substituted plinabulin compound and a preparation method and application of the polymorphism, specifically polymorphism of (3Z,6Z)-3-benzylidene-6-((5-tert-butyl-1H-imidazole-4-yl) deuterium-substituted methylene)piperazidine-2,5-diketone and preparation method and application of the polymorphism. Based on (3Z,6Z)-3-benzylidene-6-((5-tert-butyl-1H-imidazole-4-yl) deuterium-substituted methylene)piperazidine-2,5-diketone, the polymorphism of (3Z,6Z)-3-benzylidene-6-((5-tert-butyl-1H-imidazole-4-yl) deuterium-substituted methylene)piperazidine-2,5-diketone is researched, and alpha, beta, gamma, delta, and epsilon crystal forms are found. The polymorphism and preparation method and application are of great significance on research of pharmaceutical polymorphism and screening of a crystal form with excellent pharmaceutical effect, and alpha, beta, gamma, delta, and epsilon crystal forms are fully developed and applied with respect to curative effect and dosage form selection of antitumor drugs.