As　a　next-generation　digital　microfluidic　biochip　(DMFB)　platform,　micro-electrode-dot　-array　(MEDA)　play　an　important　role　in　the　biochemical　industry　with　more　advanced　fluidic　manipulation　and　higher　reliability.　As　the　MEDA　biochip　market　has　grown　by　leaps　and　bounds,　there　are　still　some　issues　with　MEDA　that　have　not　kept　pace　in　terms　of　security.　One　of　them　is　that　an　attacker　of　a　MEDA　biochip　can　change　the　protocol　parameters　by　tampering　with　the　actuation　sequence　(AS)　in　the　field　before　performing　the　bioassay,　which　can　lead　to　catastrophic　consequences.　The　attackers　could　be　untrustworthy　third　parties　introduced　by　the　distributed　supply　chain　and　malicious　remote　parties　at　the　time　of　cyber-physical　MEDA　biochip　deployment.　In　this　paper,　a　two-layer　authentication　solution　for　AS　tampering　attack　detection　is　proposed,　based　on　SHA-256　hash　function　to　ensure　the　security　of　the　authentication　process　and　at　the　same　time,　it　is　able　to　detect　and　locate　the　tampered　AS　with　simplicity,　security　and　efficiency.　Experiments　demonstrate　the　obvious　advantages　in　security,　performance　and　applicability　within　a　controllable　time　complexity.　©　2023　ACM.