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The volatile organic compounds emitted by plants significantly impact the atmospheric environment. The impacts of drought stress on the biogenic volatile organic compound (BVOC) emissions of plants are still under debate. In this study, the effects of two drought–rehydration cycle groups with different durations on isoprene emissions from Populus nigra (black poplar) seedlings were studied. The P. nigra seedlings were placed in a chamber that controlled the soil water content, radiation, and temperature. The daily emissions of isoprene and physiological parameters were measured. The emission rates of isoprene (Fiso) reached the maximum on the third day (D3), increasing by 58.0% and 64.2% compared with the controlled groups, respectively, and then Fiso significantly decreased. Photosynthesis decreased by 34.2% and 21.6% in D3 in the first and second groups, respectively. After rehydration, Fiso and photosynthesis recovered fully in two groups. However, Fiso showed distinct inconsistencies in two groups, and the recovery rates of Fiso in the second drought group were slower than the recovery rates of Fiso in the first groups. The response of BVOC emissions during the drought-rehydration cycle was classified into three phases, including stimulated, inhibited, and restored after rehydration. The emission pattern of isoprene indicated that isoprene played an important role in the response of plants to drought stress. A drought–rehydration model was constructed, which indicated the regularity of BVOC emissions in the drought–rehydration cycle. BVOC emissions were extremely sensitive to drought, especially during droughts of short duration. Parameters in computational models related to BVOC emissions of plants under drought stress should be continuously improved.