Quercus aquifolioides is one of the most representative broad-leaved plants in Qinghai-Tibet Plateau with important ecological status. So far, understanding how to quickly estimate the chlorophyll content of plants in plateau areas is still an urgent problem. Field Spec 3 spectrometer was used to measure hyperspectral reflectance data of Quercus aquifolioides leaves at different altitudes, and CCI (chlorophyll relative content) of corresponding leaves was measured by a chlorophyll meter. The correlation and univariate linear fitting analysis techniques were used to establish their relationship models. The results showed that: (1) Chlorophyll relative content of Quercus aquifolioides, under different altitude gradients, were significantly different. From 2905 m to 3500 m, chlorophyll relative content increased first and then decreased. Altitude 3300 m was the most suitable growth area. (2) In 350~550 nm, the spectral reflectance was 3500 m > 3300 m > 2905 m. In 750~1100 nm, the spectral reflectivity was 2905 m > 3500 m > 3300 m. (3) There were 4 main reflection peaks and 5 main absorption valleys in the leaf surface spectral reflection curve. While, 750~1400 nm was the sensitive range of leaf spectral response of Quercus aquifolioides. (4) The red edge position and red valley position moved to short wave direction with the increase of altitude, while the yellow edge position and green peak position moved to long wave direction first and then to short wave direction. (5) The correlation curve between the original spectrum and the CCI value was the best between the wavelengths 509~650 nm. The correlation between the first derivative spectrum and CCI value was the best and most stable at 450~500 nm. The green peak reflectance was most sensitive to the relative chlorophyll content of Quercus aquifolioides. The estimation model R² of green peak reflectance was the highest (y = 206.98e^−10.85x, R² = 0.8523), and the prediction accuracy was 95.85%. The research results can provide some technical and theoretical support for the protection of natural Quercus aquifolioides forests in Tibet.