Display omitted •A new freely available ImageJ-based plugin called DiAna is presented.•DiAna proposes two 3D procedures for image segmentation.•DiAna proposes 3D automated object-based co-localization analysis.•We introduce a novel method for statistical significance of object-based co-localization.•DiAna has extended functions for 3D distance analysis and 3D measurements. We present a new plugin for ImageJ called DiAna, for Distance Analysis, which comes with a user-friendly interface. DiAna proposes robust and accurate 3D segmentation for object extraction. The plugin performs automated object-based co-localization and distance analysis. DiAna offers an in-depth analysis of co-localization between objects and retrieves 3D measurements including co-localizing volumes and surfaces of contact. It also computes the distribution of distances between objects in 3D. With DiAna, we furthermore introduce an original method, which allows for estimating the statistical significance of object co-localization. DiAna offers a complete and intuitive 3D image analysis tool for biologists.