Real-time　imaging　of　reactive　oxygen　species　(ROS)　during　cisplatin　chemotherapy　of　cancer　is　imperative　to　fully　reveal　their　functions　in　the　biological　response　to　cisplatin.　Currently,　using　a　bioluminescent　probe　for　real-time　imaging　of　a　specific　ROS　in　vivo　during　cisplatin　chemotherapy　has　not　been　achieved.　Herein,　three　bioluminescent　probes,　F　Probe,　N　Probe　and　P　Probe　were　synthesized　for　real-time　imaging　of　the　primary　ROS,　O2•−.　They　all　consisted　of　a　bioluminescent　emitter　D-luciferin　(D-LH2)　and　an　O2•−-recognition　group,　and　their　bioluminescent　signal　could　be　turned　on　in　response　to　O2•−.　In　vitro　results　indicated　that　P　Probe　was　the　most　suitable　one　among　the　three　probes　for　detection　of　O2•−,　with　high　sensitivity,　excellent　selectivity　and　stability.　P　Probe　was　then　successfully　applied　for　real-time　imaging　of　O2•−　in　both　cancer　cells　and　tumors　during　cisplatin　chemotherapy.　The　imaging　results　demonstrated　that　O2•−　amount　in　cancer　cells　increased　with　the　increasing　dose　of　cisplatin,　and　that　cisplatin-induced　upregulation　of　O2•−　level　in　cancer　cells　was　upstream　of　the　cancer-killing　pathway　of　cisplatin.　We　envision　that　P　Probe　may　serve　as　an　elucidative　tool　to　further　explore　the　role　of　O2•−　in　cisplatin　chemotherapy.　©　2022