Integrated treatment using imaging technology to monitor biological processes for the precise treatment and diagnosis of diseases to improve treatment outcomes is becoming a hot topic. Accordingly, perylene diimide (PDI) has excellent photothermal conversion and photostability, which can be used as a good material for disease treatment and diagnosis. Herein, we review the latest research progress on the real-time diagnosis of related diseases based on perylene diimide probes in the aspects of bioimaging, detection of biomarkers and determination of the pH in living cells. Furthermore, perylene diimide-based multifunctional nano-delivery systems are particularly emphasized, showing great therapeutic potential in the field of image-guided combination therapy in tumor therapy. Finally, the great opportunities and challenges still faced by perylene diimide before entering the clinic are comprehensively analyzed.