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Advantages such as strong signal strength, resistance to photobleaching, tunable fluorescence emissions, high sensitivity, and biocompatibility are the driving forces for the application of fluorescent nanoparticles (FNPs) in cancer diagnosis and therapy. In addition, the large surface area and easy modification of FNPs provide a platform for the design of multifunctional nanoparticles (MFNPs) for tumor targeting, diagnosis, and treatment. In order to obtain better targeting and therapeutic effects, it is necessary to understand the properties and targeting mechanisms of FNPs, which are the foundation and play a key role in the targeting design of nanoparticles (NPs). Widely accepted and applied targeting mechanisms such as enhanced permeability and retention (EPR) effect, active targeting, and tumor microenvironment (TME) targeting are summarized here. Additionally, a freshly discovered targeting mechanism is introduced, termed cell membrane permeability targeting (CMPT), which improves the tumorâ targeting rate from less than 5% of the EPR effect to more than 50%. A new design strategy is also summarized, which is promising for future clinical targeting NPs/nanomedicines design. The targeting mechanism and design strategy will inspire new insights and thoughts on targeting design and will speed up precision medicine and contribute to cancer therapy and early diagnosis., J.H., C.L., L.D., and Y.H. contributed equally to this work. This work was been supported by the National Natural Science Foundation of China (Nos. 21371115, 11025526, 21671131, 11875185), the Shanghai University-Universal Medical Imaging Diagnostic Research Foundation (19H00100), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13078) and the Portuguese Foundation for Science and Technology (IF/00347/2015).