SUMMARY: Computational methods that track single cells and quantify fluorescent biosensors in time-lapse microscopy images have revolutionized our approach in studying the molecular control of cellular decisions. One barrier that limits the adoption of single-cell analysis in biomedical research is the lack of efficient methods to robustly track single cells over cell division events. Here, we developed an application that automatically tracks and assigns mother-daughter relationships of single cells. By incorporating cell cycle information from a well-established fluorescent cell cycle reporter, we associate mitosis relationships enabling high fidelity long-term single-cell tracking. This was achieved by integrating a deep-learning-based fluorescent proliferative cell nuclear antigen signal instance segmentation module with a cell tracking and cell cycle resolving pipeline. The application offers a user-friendly interface and extensible APIs for customized cell cycle analysis and manual correction for various imaging configurations. AVAILABILITY AND IMPLEMENTATION: pcnaDeep is an open-source Python application under the Apache 2.0 licence. The source code, documentation and tutorials are available at https://github.com/chan-labsite/PCNAdeep. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.