Abstract: To explore the mechanism of allelopathy on the key enzymes of seed germination, the influences of various concentrations of water extract from Eupatorium adenophorum on the activities of six enzymes, namely peroxidase, catalase, acid phosphatase, cellulase, α-amylase, and β-amylase, during the seed germination of Pinus yunnanensis were examined using the petri dish filter paper method. Moreover, the relationship between seed germination and enzyme activities was analyzed. The findings indicated that different concentrations of the water extract had diverse effects on the seed germination and enzyme activities of Pinus yunnanensis. When the concentration of the water extract was 0.005 g/mL, the seed germination rate, germination potential, and the activities of the six enzymes of Pinus yunnanensis were higher than those of the control. The inhibitory effect strengthened as the concentration of the water extract increased. The seed germination rate was positively correlated with the activities of catalase, acid phosphatase, and cellulase, and also positively correlated with the activities of the other three enzymes. The seed germination and natural regeneration of Pinus yunnanensis could be impacted by Eupatorium adenophorum through the influence of enzyme activity.