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Three structure and operation parameters which had effect on vertical axial-flow corn single panicle thresher were optimized. The quadratic rotary orthogonal experiments were carried out. Taking linear velocity of threshing cylinder, negative pressure value of fan, moisture content of cluster as effect factors, and broken rate, impurities, loss of entrapped grains, gettering drift loss as objective function, the structure size and operation parameters were experimented and optimized design. The result showed that when the linear velocity of threshing cylinder was 4.6~5.5m/s, the moisture content of cluster was 14.4%~15.9%, the negative pressure value of fan was 6.3MPa, the broken rate was less than 1.1%, impurities rate was less than 0.86%, loss rate of entrapped grains was less than 1.1%, and gettering drift loss rate was less than 0.44%. This experiment’s analysis could provide theory basis for the optimization design and operation parameter selection for the vertical axial single panicle threshing corn product.