Nanomedicines overcome the pharmacokinetic limitations of traditional drug formulations and have promising prospect in cancer treatment. However, nanomedicine delivery in vivo is still facing challenges from the complex physiological environment. For the purpose of effective tumor therapy, they should be designed to guarantee the five features principle, including long blood circulation, efficient tumor accu-mulation, deep matrix penetration, enhanced cell internalization and accurate drug release. To ensure the excellent performance of the designed nanomedicine, it would be better to monitor the drug delivery process as well as the therapeutic effects by real-time imaging. In this review, we summarize strategies in developing nanomedicines for efficiently meeting the five features of drug delivery, and the role of sev-eral imaging modalities (fluorescent imaging (FL), magnetic resonance imaging (MRI), computed tomog-raphy (CT), photoacoustic imaging (PAI), positron emission tomography (PET), and electron microscopy) in tracing drug delivery and therapeutic effect in vivo based on five features principle.(c) 2022 Elsevier B.V. All rights reserved.