Selenium is an important and essential trace element to living systems. In the article, two methods of instrumental neutron activation analysis and hydride generation-atomic fluorescence spectrometry were applied to determine Se in biological samples and the accuracy was evaluated by several reference materials. The subcellular distribution of selenium in human liver samples, which were obtained from normal subjects who had an accidental death, was investigated by differential centrifugation combined with INAA. Selenium was mainly enriched in mitochondria, nuclei, and cytosol. Almost half of the total Se content existed in nuclei as a result of the large amount in liver and the high Se concentration. Generally, the highest Se concentration in the mitochondrial fractions of each liver sample suggested that Se had important functions in this liver component.