In this study, to find out the optimal lysis buffer for dissociating cell nucleus, five common lysis buffers (Tris?MgCl2, LB01, WPB, Otto’s, Galbraith’s) were tested on the cell nucleus of Alsophila spinulosa leaves by use of Gnetum montanum as internal standard, and, the genome size of A. spinulosa was calculated according to the multiple relationships of peak values of A. spinulosa and Gnetum montanum using flow cytometry. The results showed that the WPB was the most appropriate lysis buffer to dissociate A. spinulosa cell nucleus and the genome size of A. spinulosa was determined to be 6 003.25 Mb, i.e. 2C DNA content was 6.138 pg.