A new segmentation method for segmenting the scalp, skull, cerebrospinal fluid (CSF) layer, grey matter and white matter in MRI of mouse brain was proposed. The method is based on the threshold, K-means Clustering and Narrow Band Level Set methods. Firstly, the threshold for skull segmentation is obtained by simple interaction. Secondly, skull segmentation using threshold methods combined with dilation and erosion and the skull upper surface is extracted. Thirdly, brain is extracted using narrow band level set method and scalp is obtained by minus other tissues. In the next step, brain is segmented by fuzzy c-means clustering. Finally, the 3D segmentation result is reconstructed.