Carbon and nitrogen stable isotopes of herbarium specimens have been used by ecologists to study long-history pollution and climate change. However, whether the preparation and preservation procedure impact isotopic signatures of herbarium specimens remain unclear. We tested the effect of herbarium on three submerged macrophyte species, and found the variations by both preparation and preservation processes. The press procedure significantly reduced carbon stable isotopes of submerged macrophytes for P. malaianus by 1.78, M. spicatum by 0.98 and C. demersum by 0.72. The press procedure reduced C. demersum nitrogen stable isotopes by 1.1. The soak procedure did not affect nitrogen stable isotopes of submerged macrophytes, but increased carbon stable isotopes of M. spicatum and P. malaianus by 1.00 and 0.52, separately. Carbon stable isotopes of C. demersum increased less than 0.08. Preparing procedures had larger effect on carbon isotope than nitrogen isotope. These influences were highly variable between species and times. The results suggest that if the interested stable isotopic variations are within 3, herbarium specimens are not suitable samples for the relevant studies without the experimental corrections.