Abstract：To discuss the effect of root canal seal achieved by the irradiation of Er,Cr:YSGG laser and to provide the important experimental basis for clinical application of Er,Cr:YSGG laser. Eighty freshly single-rooted extracted human teeth were randomly divided into four groups of twenty each. The anatomical crowns were removed and the remaining roots were prepared endodontically with conventional methods. Laser irradiation was performed at different output powers with water spray cooling. Specimens were evaluated morphologically by using scanning electron microscopy. After root canal obturation, the effect of root canal seal achieved by Er,Cr:YSGG laser was evaluated with microleakage measurement. One-way analysis of variance and Newman-Keuls test were used to determine the statistical differences between various groups. A value of P<0.05 was considered to be significant. The results of morphological observation showed that Er,Cr:YSGG laser irradiation may abolish smear layer and debris on the root canal walls effectively. Little or no fusing and carbonization was observed. In the groups, the effect of laser irradiation at 3 W was the best. Furthermore, microleakage measurement showed that compared with the negative control, the depth of dye penetration with the irradiation of laser was decreased significantly (P<0.05); but in the laser irradiation groups, the statistical differences were not significant (P>0.05). Therefore, the utilizing of Er,Cr :YSGG laser in root canal preparation can clean up root canal ideally and promote the effect of root canal seal, which may be applied as a therapeutic tool for root canal therapy.