The V3D system provides three-dimensional (3D) visualization of gigabyte-sized microscopy image stacks in real time on current laptops and desktops. Combined with highly ergonomic features for selecting an X, Y, Z location of an image directly in 3D space and for visualizing overlays of a variety of surface objects, V3D streamlines the on-line analysis, measurement, and proofreading of complicated image patterns. V3D is cross-platform and can be enhanced by plug-ins. We built V3D-Neuron on top of V3D to reconstruct complex 3D neuronal structures from large brain images. V3D-Neuron enables us to precisely digitize the morphology of a single neuron in a fruit fly brain in minutes, with about 17-fold improvement in reliability and 10-fold savings in time compared to other neuron reconstruction tools. Using V3D-Neuron, we demonstrated the feasibility of building a 3D digital atlas of neurite tracts in the fruit fly brain.