In　this　study,　tea　polyphenols　(TPs)　was　first　extracted　from　tea　waste　by　superheated　steam　(SS)　pretreated　ultrasonic-assisted　hydrothermal　extraction　(UAH).　The　optimized　strategy　presented　extracts　with　the　extraction　yield　up　to　21.19%　with　a　significantly　higher　antioxidant　ability,　compared　with　the　one　without　SS　pretreatment.　Further　investigation　proved　that　the　SS　suppressed　the　polyphenol　oxidase　activity　of　the　TPs　extract.　The　ability　to　scavenge　the　free　radicals　were　compared　in　mouse　liver　mitochondria.　Mitochondrial　swelling,　mitochondrial　membrane　potential　(MMP),　cardiolipin　peroxidation,　and　respiratory　chain　complex　(RCC)　I–V　activities　were　also　evaluated　as　the　index　of　the　mitochondrial　oxidative　damage.　The　study　supports　evidence　that　the　TPs　extract　exhibited　significant　protection　against　oxidative　damage　on　mitochondrial.　Furthermore,　the　effect　of　TPs　on　antioxidant　ability　in　zebrafish　embryo　was　evaluated.　After　TPs　pretreatment　for　1 day,　zebrafish　embryos　showed　a　significantly　higher　survival　rate　as　well　as　heart　rate　when　facing　the　oxidative　stress.　Practical　applications:　Polyphenols　from　tea　leaves　have　been　viewed　as　an　antioxidant　additive　in　food,　mainly　due　to　the　ability　of　scavenging　free　radicals　and　reactive　oxygen　species.　The　results　of　this　study　suggest　that　the　SS　pretreatment　could　be　used　as　an　efficient　method　to　extract　TPs　from　the　tea　waste　for　the　prevention　of　oxidative　damage　in　the　mouse　liver　mitochondria　and　zebrafish　embryos.　©　2019　Wiley　Periodicals,　Inc.