The integration of a drug and polyethylene glycol (PEG) into an amphiphilic polymer to form self-assembled micelles with high drug loading for self-delivery to tumors is recently reported, which has showed high clinical availability. However, the effect of PEG chain length on cell internalization efficiency and tumor targeting efficiency in this PEG-drug conjugate micelle self-delivery system has yet to be studied. Herein, pyropheophorbide-a (PhA) was used as a model drug and a fluorescent tracer to synthesize amphiphilic polymers with varying PEG chain lengths, which self-assembled into PhA-PEG₂₀₀₀ NPs and PhA-PEG₅₀₀₀ NPs with high drug loading (21.2% and 9.7%, respectively, according to calculation). The results showed that PhA-PEG₂₀₀₀ NPs had higher cell internalization in vitro than PhA-PEG₅₀₀₀ NPs, but much lower tumor accumulation in vivo than PhA-PEG₅₀₀₀ NPs. This was attributed to the longer in vivo blood circulation time of the latter. Furthermore, the in vitro and in vivo examinations revealed negligible side effects, indicating the intrinsic safety of the micelles. This study provides insight into the influence of PEG chain length on biodistribution of organs and tumor accumulation after intravenous administration of PEG-drug conjugate micelle and guides the design of drug incorporated amphiphilic polymer-based micelles.