A　new　electrocatalytic　biosensor　(MOF-74(Cu)　NS-CC)　based　on　the　in　situ　deposition　of　MOF-74(Cu)　nanosheet　on　carbon　cloth　via　a　bottom-up　synthetic　approach　in　a　glass　tube　was　developed.　The　electrocatalytic　activity　of　the　deposited　MOF-74(Cu)　NS　was　demonstrated　in　the　oxidation　of　glucose　to　gluconate　under　alkaline　conditions.　The　results　revealed　that　the　proposed　method　of　in　situ　formation　of　MOF-74(Cu)　NS　onto　a　carbon　cloth　surface　in　a　multi-layer　solution　is　capable　to　generate　a　stable　MOF-74(Cu)　NS-CC　electrode　with　excellent　sensing　performance.　When　the　as-synthesized　MOF-74(Cu)　NS-CC　was　applied　directly　as　the　working　electrode　for　glucose　sensing,　it　showed　much　higher　conductivity　and　redox　activity　than　MOF-74(Cu)　NS-GCE.　With　the　potential　applied　at　0.55　V　(vs.　Ag/AgCl),　this　new　electrocatalytic　biosensor　exhibits　an　excellent　linear　relationship　between　current　density　and　concentration　of　glucose.　Moreover,　a　wide　linear　range　of　detection　(1.0　to　1000　mu　M)　was　observed.　The　limit　of　detection　was　found　to　be　0.41　mu　M　(S/N　=　3).　The　response　sensitivity　is　3.35　mA　mM(-1)　cm(-2)　when　the　concentration　of　glucose　is　in　the　range　1-100　mu　M　and　3.81　mA　mM(-1)　cm(-2)　for　the　100-1000　mu　M　concentration　range.　This　study　provides　a　low-cost,　easy　to　prepare,　and　reproducible　methodology　for　the　synthesis　of　highly　redox-active　nanomaterials　based　on　the　in　situ　formation　of　two-dimensional　MOF-74(Cu)　NS　for　the　development　of　new　electrocatalytic　biosensors.　Graphical　abstract