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Segmenting individual neurons from a large number of noisy raw images is the first step in building a comprehensive map of neuron-to-neuron connections for predicting information flow in the brain. Thousands of fluorescence-labeled brain neurons have been imaged. However, mapping a complete connectome remains challenging because imaged neurons are often entangled and manual segmentation of a large population of single neurons is laborious and prone to bias. In this study, we report an automatic algorithm, NeuroRetriever, for unbiased large-scale segmentation of confocal fluorescence images of single neurons in the adult Drosophila brain. NeuroRetriever uses a high-dynamic-range thresholding method to segment three-dimensional morphology of single neurons based on branch-specific structural features. Applying NeuroRetriever to automatically segment single neurons in 22,037 raw brain images, we successfully retrieved 28,125 individual neurons validated by human segmentation. Thus, automated NeuroRetriever will greatly accelerate 3D reconstruction of the single neurons for constructing the complete connectomes.