PURPOSE: A positive circumferential resection margin (CRM) for oesophageal and gastric carcinoma is associated with local recurrence and poorer long-term survival. Diffuse reflectance spectroscopy (DRS) is a non-invasive technology able to distinguish tissue type based on spectral data. The aim of this study was to develop a deep learning-based method for DRS probe detection and tracking to aid classification of tumour and non-tumour gastrointestinal (GI) tissue in real time. METHODS: Data collected from both ex vivo human tissue specimen and sold tissue phantoms were used for the training and retrospective validation of the developed neural network framework. Specifically, a neural network based on the You Only Look Once (YOLO) v5 network was developed to accurately detect and track the tip of the DRS probe on video data acquired during an ex vivo clinical study. RESULTS: Different metrics were used to analyse the performance of the proposed probe detection and tracking framework, such as precision, recall, mAP 0.5, and Euclidean distance. Overall, the developed framework achieved a 93% precision at 23 FPS for probe detection, while the average Euclidean distance error was 4.90 pixels. CONCLUSION: The use of a deep learning approach for markerless DRS probe detection and tracking system could pave the way for real-time classification of GI tissue to aid margin assessment in cancer resection surgery and has potential to be applied in routine surgical practice.