Fluorescence in situ hybridization (FISH) technique widely used in cancer diagnosis is based on displaying chromosomal regions as FISH signals by staining with specific dyes. In this study, a new multi-level thresholding based FISH signal segmentation method is proposed for images produced by FISH technique. Cell nuclei are segmented on images, that are grabbed from fluorescence microcopes at high resolution, with adaptive thresholding, distance transform and watershed methods. FISH signals falling in cell boundaries are detected by applying multi-level thresholding and morphological post processes thanks to proposed segmentation method. It is observed that the detection rate of the proposed method on 49 FISH images taken from real patients, are higher than other widely used techniques in the literature.