Abstract: The orthogonal experiment design was adopted to select satisfactory method for intact Cunninghamia lanceolata cell nuclei suspension preparation. The intact cell nuclei suspension of Cunninghamia lanceolata with less cell fragments and joints could be obtained through chemical dissociation, filtration, centrifugation, and refiltration. The experiments showed that the optimal dissociation conditions were taking WPB as dissociation solution, 4hr of dissociation, and 800r/min centrifugation for 8min. Filtration with 50μm filter cotton could significantly reduce the cell fragments and joints and promote the purity of the intact cell nuclei suspension.