Abstract: AimTo determine the location and relative quantity of coumarins in the stem of Dendrobium thyrsiflorum Rchb. f., and to provide a scientific basis for evaluating and utilizing the famous medicinal plant. MethodsThe stems of one, two and three years old, separately, were collected in February. Location and relative quantity of coumarins in the top, middle and basal parts of each stem sample were determined by using laser scanning confocal microscopy (LSCM). ANOVA and Tukey′s test were employed in the statistical analysis. ResultsThe coumarins located mainly in vascular bundles, especially on the walls of the outer fiber cells. There was significant difference or great significant difference exist in every part of the stem from different ages. The values of significant difference were as follows: basal part P=0.004(<0.01); middle part P=0.009(<0.01); top part P=0.036(<0.05). ConclusionTwo years old stem could be the best choice when collecting Dendrobium thyrsiflorum in February.