This study aimed to prepare mitoxantrone thermosensitive liposome (MTX-TSL) to enhance the targeting capability of liposomes and thus improve the therapeutic effect of the drug on prostate cancer. MTX-TSL were prepared using the thin-film hydration method. A single-factor experiment was conducted to optimize the formulation process, and the liposome quality was assessed alongside short-term stability testing. The in vitro efficacy of MTX-TSL was evaluated using RM-1 prostate cancer cell inhibition assays. In vivo experiments were conducted on BDF1 mice inoculated with RM-1 cells to assess the tissue distribution and anticancer activity of MTX-TSL. Quality assessments of MTX-TSL revealed a pH of 6.53 +/- 0.02, osmotic pressure of 309 +/- 3 mOsmol/Kg, particle size of 100.10 +/- 1.50 nm, and encapsulation efficiency of 98.41% +/- 0.23%. Stability tests showed no major quality changes for liposome suspensions stored at 2-8 degrees C for 2 months. In vitro release studies showed that MTX-TSL exhibited good thermosensitive properties. Experiments performed on BDF1 mice indicated that initiating hyperthermia before drug administration was beneficial for drug accumulation in tumor tissue and that MTX-TSL outperformed free drugs in suppressing tumor growth when combined with appropriate hyperthermia. MTX-TSL can effectively inhibit tumor growth while increasing the drug's therapeutic index.