Crude protein (CP), crude fat (CFA) and crude fiber (CFI) are key indicators for evaluation of the quality and feeding value of pasture. Hence, identification of these biological contents is an essential practice for animal husbandry. As current approaches to pasture quality estimation are time-consuming and costly, and even generate hazardous waste, a real-time and non-destructive method is therefore developed in this study using pasture canopy hyperspectral data. A field campaign was carried out in August 2013 around Qinghai Lake in order to obtain field spectral properties of 19 types of natural pasture using the ASD Field Spec 3, a field spectrometer that works in the optical region (350-2 500 nm) of the electromagnetic spectrum. In additional to the spectral data, pasture samples were also collected from the field and examined in laboratory to measure the relative concentration of CP (%), CFA (%) and CFI (%). After spectral denoising and smoothing, the relationship of pasture quality parameters with the reflectance spectrum, the first derivatives of reflectance (FDR), band ratio and the wavelet coefficients (WCs) was analyzed respectively. The concentration of CP, CFA and CFI of pasture was found closely correlated with FDR with wavebands centered at 424, 1 668, and 918 nm as well as with the low-scale (scale = 2, 4) Morlet, Coiflets and Gassian WCs. Accordingly, the linear, exponential, and polynomial equations between each pasture variable and FDR or WCs were developed. Validation of the developed equations indicated that the polynomial model with an independent variable of Coiflets WCs (scale = 4, wavelength =1 209 nm), the polynomial model with an independent variable of FDR, and the exponential model with an independent variable of FDR were the optimal model for prediction of concentration of CP, CFA and CFI of pasture, respectively. The R2 of the pasture quality estimation models was between 0.646 and 0.762 at the 0.01 significance level. Results suggest that the first derivatives or the wavelet coefficients of hyperspectral reflectance in visible and near-infrared regions can be used for pasture quality estimation, and that it will provide a basis for real-time prediction of pasture quality using remote sensing techniques.