Eotetranychus sexmaculatus Riley is an important pest mite on rubber trees, it causes damage to rubber leaves, causing localized chlorotic spots to spread to the entire leaf, resulting in severe yellowing. Severe infestations result in massive leaf drop, disrupting normal tapping operations and leading to yield losses. However, the changes in physiological and biochemical indicators of rubber tree leaves after mite damage, and the relationship with mite population density and infestation duration, remain unclear. This study aimed to reveal the effects of varying mite densities and infestation durations on physiological and biochemical traits in rubber tree leaves, and to elucidate the defense mechanisms of rubber trees, thereby providing a theoretical basis for mite monitoring and early warning and precise prevention and control of the mite. In this study, the effects of malondialdehyde (MDA), soluble sugars, soluble proteins and protective enzymes (SOD, POD, CAT) activities on the leaves of rubber trees with different infestation times were determined on the rubber potted seedlings of the same length of Thermo Scientific 7-33-97 rubber seedlings inoculated with different mite densities in the greenhouses. The two-way analysis of variance (ANOVA) was used to reveal the interaction between mite population density and infestation time. Regarding membrane lipid peroxidation damage, both mite population density and infestation duration significantly influenced MDA content, exhibiting an initial increase followed by a decrease as infestation time prolonged. MDA levels peaked at (73.91±1.89)nmol/g after 15 days of treatment with 40 mites per leaf, representing a 43.15% increase compared to the control. MDA levels began to decline to (65.09±0.29)nmol/g after 20 days. Both soluble sugar and soluble protein contents decreased with increasing mite density and duration of infestation. At a mite density of 40 mites/leaf with 20 days of continuous feeding, soluble sugar and soluble protein contents reached the lowest values at (34.42±1.43)mg/g and (17.74±0.63)mg/g, respectively, representing significant decreases of 44.20% and 45.30% compared to the control. In response to mite-induced oxidative stress, the protective enzyme activities (SOD, POD, CAT) in rubber tree leaves generally showed a significant upward trend during the early stages of mite infestation across different mite density and duration treatments. Enzyme activity peaks occurred earlier under high mite density treatments (30 mites/leaf and 40 mites/leaf). Specifically, SOD activity peaked at (13 086.92±613.39)mg/g after 5 days of 40 mites/leaf infestation, representing a 91.74% increase compared to the control. POD activity reached minor peaks at 10 days post-infestation in both 30 and 40 mites/leaf treatments (6293.13±80.75)U/(min·g) and (6655.54±51.44)U/(min·g), respectively, followed by a decline before rising again. CAT activity peaked at 10 days in the 40 mites/leaf treatment (165.77±0.41)µmoL/(min·g), representing a 62.74% increase compared to the control. Interaction analysis revealed that the interaction between mite density and damage duration significantly affected soluble sugar content (F=21.296, P<0.001), soluble protein (F=17.782, P<0.001), and significantly affected SOD (F=20.252, P<0.001), POD (F=9.821, P<0.001), and CAT (F=145.095, P<0.001) activities. Both the mite population density and the duration of damage inflicted by the E. sexmaculatus leaf mite can influence the physiological and biochemical indicators in rubber tree leaves, including malondialdehyde, soluble sugars, soluble proteins, and protective enzymes (SOD, POD, CAT). Furthermore, a significant interaction exists between mite population density and duration of damage (P<0.001). This confirms that stress intensity and duration do not act independently, but they jointly determine the physiological damage degree, nutritional status, and antioxidant defense efficiency of rubber tree leaves through a complex synergistic mechanism.