As　a　form　of　cold　atmospheric　plasma　application,　plasma-treated　solutions　have　shown　promise　in　tumor　therapy.　Especially　for　the　treatment　of　intracavitary　tumors,　plasma-treated　solutions　combined　with　traditional　perfusion　therapy　have　strong　application　potential.　Here,　we　assessed　the　antitumor　effects　of　the　plasma-treated　medium　combined　with　hyperthermia　on　three-dimensional　bladder　tumor　spheroids　in　vitro.　We　found　that　the　combined　treatment　of　plasma-treated　medium　and　hyperthermia　(43　degrees　C)　presented　better　antitumor　effects　than　a　single　treatment.　Further　research　showed　that　synchronous　hyperthermia　can　promote　the　penetration　of　plasma-generated　reactive　oxygen　species　from　the　plasma-treated　medium　into　cancer　cells.　Our　results　suggest　that　hyperthermic　perfusion　therapy　using　plasma-treated　solutions　may　serve　as　a　novel　and　valuable　therapeutic　strategy　for　bladder　cancer.