Abstract: To discuss and optimize the methods of estimating nuclear DNA content of medicinal plants by flow cytometry (FCM), fresh leaves, younger leaves and dry leaves by silica were taken as samples and Otto’s buffer and LB01 buffer were used to estimate the C-value of Scutellaria baicalensis Georgi by FCM. The results showed that better results were easily and accurately gotten with relatively concentrated scatterplot, less debris, more simplicity to take gates and lower CV when using Otto’s buffer with two-steps method to extract, prepare and store under low temperature with the concentration of propidium iodide (PI) at 0.02 mg/mL , compared to that by using LB01 buffer. The results of different materials demonstrated that both fresh leaves and younger leaves could get better quality suspension of nuclei by Otto’s and generated a larger amount of nuclei and more concentrated particles in scatterplots, which would help take gates and estimate C-value more accurately. On the contrary, younger leaves would lead to a large number of centralized nuclei, which was unable to take gates and estimate C-value accurately. At last, S. baicalensis was taken as example to optimize the methods of estimating nuclear DNA content of medicinal plants, and the C-value of S. baicalensis was first estimated successfully as 1C= 0.58 pg, and the genome size was 562 Mbp by FCM. These results will thus provide theoretical basis for the research of recognizing the genetic background of species and changing disciplines of genome size in phylogeny and the genetic evaluation, conservation and development of medicinal plant resources of Scutellaria.