In this paper, we propose a highly automated 3-D centerline extraction tool to assist 3-D microscopy image analysis to extract morphological features of the neuronal structures. For this project, the most difficult part is that, some axons are overlapping to each other that the boundaries distinguishing them are hardly visible. Our approach combines 3-D Dynamic Programming (DP) technique and marker controlled watershed algorithm to solve this problem. The approach consists of tracking and updating along the navigation directions of multiple axons simultaneously. The experimental results show that, the proposed method can rapidly and accurately extract multiple axon centerlines and can handle complicated axon structures such as cross-over sections and overlapping objects.