Sargent’s cherry trees (Prunus sargentiiRehder) are widely planted as an ornamental, climate change-sensing species. This study investigated changes in the soil moisture content, fresh weight, photosynthesis and chlorophyll fluorescence properties, and the chlorophyll and proline content of four-year-old P. sargentii seedlings after 30 days of drought stress. In the trees subjected to drought stress treatment, soil moisture content decreased, and the fresh weight of the aboveground part of the plant decreased. However, there was no significant difference in the root growth of the dried plants. Among the photosynthesis parameters, Pn MAX, E and gs showed a significant (p < 0.001) decrease after 15 days in dry-stressed seedlings, but there was no difference between treatments in WUE until 20 days, and there was a significant (p < 0.001) difference after 24 days. Chlorophyll fluorescence parameters, Fv/Fm, ΦPSII, Rfd, NPQ, and Pn MAX, also increased after 10 days in dry-stressed seedlings, but these changes did not reach statistical significance compared to the control treatment. These results may suggest that drought stress highly correlates with photosynthesis and chlorophyll fluorescence parameters. Chlorophyll content also significantly decreased in the seedlings under drought stress compared with the control treatment. The proline content decreased until the 10th day of drought stress treatment and increased after the 15th day, showing an increase of 10.9% on the 15th day and 57.1% on the 30th day, compared to the control treatment. These results suggest that photosynthesis, chlorophyll fluorescence parameters, and proline content can be used to evaluate drought stress in trees. The results of this study can contribute to the management of forests, such as the irrigation of trees when pore control ability and photosynthesis ability decrease.