Liposomes are potent nanocarriers to deliver chemotherapeutic drugs to tumors. However, the inefficient drug release hinders their application. Thermosensitive liposomes (TSL) can release drugs upon heat. This study aims to identify the optimum 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 (DSPE-PEG2000) concentration in stealth TSL to improve content release efficiency under mild hyperthermia (HT). TSL were prepared with DSPE-PEG2000 from 1 to 10mol%, around 80nm in size. Quenched carboxyfluorescein (CF) in aqueous phase represented encapsulated drugs. In vitro temperature/time-dependent CF release and TSL stability in serum were quantified by fluorometry. In vivo CF release in dorsal skin flap window chamber models implanted with human BLM melanoma was captured by confocal microscopy. In vitro heat triggered CF release increased with increasing DSPE-PEG2000 density. However, 6mol% and higher DSPE-PEG2000 caused CF leakage at physiological temperature. TSL with 5mol% DSPE-PEG2000 were stable at 37°C, while released 60% CF in 1min and almost 100% CF in 1h at 42°C. In vivo optical intravital imaging showed immediate massive CF release above 41°C. In conclusion, incorporation of 5mol% DSPE-PEG2000 optimized stealth TSL content release triggered by HT.