Using　sludge　obtained　from　municipal　sewage　treatment　plants,　the　response　of　viable　bacterial　populations　during　the　sludge　ozonation　process　was　investigated　by　a　combination　of　adenosine　triphosphate　(ATP)　assay　and　propidium　monoazide　(PMA)-Miseq　sequencing.　The　ATP　assay　was　first　optimized　for　application　on　sludge　samples　by　adjusting　the　sludge　solid　contents　and　reaction　time.　PMA-modified　polymerase　chain　reaction　(PCR)　was　also　optimized　by　choosing　the　suitable　final　PMA　concentration.　The　quantity　and　composition　of　viable　bacterial　populations　during　sludge　ozonation　were　further　elucidated　using　the　optimized　ATP　and　PMA-modified　PCR　methods.　The　results　indicated　that　after　the　sludge　was　exposed　to　ozone　(O-3)　at　135　mgO(3)/g　total　suspended　solids　(TSS),　the　viable　biomass　displayed　a　substantial　decrease,　with　a　reduction　rate　reaching　70.89%.　The　composition　of　viable　bacterial　communities　showed　a　faster　succession,　showing　that　an　ozone　dosage　of　114　mgO(3)/g　TSS　is　enough　to　significantly　change　the　viable　bacterial　population　structure.　Floc-forming　genera,　such　as　Zoogloea,　Ferruginibacter,　Thauera　and　Turneriella,　are　sensitive　to　ozonation,　while　the　relative　abundances　of　some　functional　bacterial　genera,　including　SM1A02,　Nitrospira　and　Candidatus　Accumulibacter,　remained　constant　or　increased　in　the　viable　bacterial　population　during　sludge　ozonation,　indicating　that　they　are　more　resistant　to　ozonation.