2D　nanosheets　(NSs)　have　been　widely　used　in　drug-related　applications.　However,　a　comprehensive　investigation　into　the　cytotoxicity　mechanism　linked　to　the　redox　activity　is　lacking.　In　this　study,　with　cytochrome　c　(Cyt　c)　as　the　model　biospecies,　the　cytotoxicity　of　2D　NSs　was　evaluated　systematically　based　on　their　redox　effect　with　microfluidic　techniques.　The　interface　interaction,　dissolution,　and　redox　effect　of　2D　NSs　on　Cyt　c　were　monitored　with　pulsed　streaming　potential　(SP)　measurement　and　capillary　electrophoresis　(CE).　The　relationship　between　the　redox　activity　of　2D　NSs　and　the　function　of　Cyt　c　was　evaluated　in　vitro　with　Hela　cells.　The　results　indicated　that　the　dissolution　and　redox　activity　of　2D　NSs　can　be　simultaneously　monitored　with　CE　under　weak　interface　interactions　and　at　low　sample　volumes.　Both　WS2　NSs　and　MoS2　NSs　can　reduce　Cyt　c　without　significant　dissolution,　with　reduction　rates　measured　at　6.24　×　10-5　M　for　WS2　NSs　and　3.76　×　10-5　M　for　MoS2　NSs.　Furthermore,　exposure　to　2D　NSs　exhibited　heightened　reducibility,　which　prompted　more　pronounced　alterations　associated　with　Cyt　c　dysfunction,　encompassing　ATP　synthesis,　modifications　in　mitochondrial　membrane　potential,　and　increased　reactive　oxygen　species　production.　These　observations　suggest　a　positive　correlation　between　the　redox　activity　of　2D　NSs　and　their　redox　toxicity　in　Hela　cells.　These　findings　provide　valuable　insight　into　the　redox　properties　of　2D　NSs　regarding　cytotoxicity　and　offer　the　possibility　to　modify　the　2D　NSs　to　reduce　their　redox　toxicity　for　clinical　applications.　©　2024　American　Chemical　Society.