Abstract: In the determination of biological samples by X-ray Fluorescence Spectrometry, sample powder pellets pressed by traditional sample preparation technique at 220-440 MPa are not compact and smooth, so the sample room of X-ray Fluorescence Spectrometry becomes contaminated by dropped sample powder, which can then influence long term stability. Biological sample powder can be pressed into smooth and compact pellets using high pressure at 1760 MPa, therefore the sample preparation reproducibility is improved to 0.1%-2.6% (RSD, n=5). A method for direct determination of 23 major and minor elements (Al, Ca, Cl, K, Mg, Na, P, S, Si, Ba, Br, Co, Cr, Cu, Fe, Mn, Ni, Pb, Rb, Sr, Ti, V and Zn) in biological samples by Wavelength Dispersion X-ray Fluorescence Spectrometry was established on this sample preparation basis. The matrix effects can be corrected by using Rh-Kα(from the X-ray tube target) Compton-scattered radiation and by using the background as the internal standard. The precision of the method is 0.1%-11.3% (RSD) for most elements and the detection limits is 0.08-140.96 μg/g. The feasibility of the proposed method was tested by analyzing several national biological standard materials; the results obtained were consistent with the certified values.