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New amphiphilic star or multi-arm block copolymers with different structures were synthesized for enabling the use of hydrophobic oxygen probe of platinum (II)-tetrakis (pentafluorophenyl) porphyrin (PtTFPP) for bioanalysis. The amphiphilic star polymers were prepared through the Atom Transfer Radical Polymerization (ATRP) method by using hydrophilic 4-arm polyethylene glycol (4-arm-PEG) as an initiator. Among the five block copolymers, P1 series (P1a, P1b, and P1c) and P3 possess fluorine-containing moieties to improve the oxygen sensitivity with its excellent capacity to dissolve and carry oxygen. A polymer P2 without fluorine units was also synthesized for comparison. The structure-property relationship was investigated. Under nitrogen atmosphere, high quantum efficiency of PtTFPP in fluorine-containing micelles could reach to 22% and long lifetime could reach to 76 μs. One kind of representative PtTFPP-containing micelles was used to detect the respiration of Escherichia coli (E. coli) JM109 and macrophage cell J774A.1 by a high throughput plate reader. In vivo hypoxic imaging of tumor-bearing mice was also achieved successfully. This study demonstrated that using well-designed fluoropolymers to load PtTFPP could achieve high oxygen sensing properties, and long lifetime, showing the great capability for further in vivo sensing and imaging.