In　tunnelling　using　tunnel　boring　machine　(TBM),　the　surrounding　rocks　are　typically　supported　by　precast　lining　segments,　pea-gravel　backfilling　and　grouting.　The　compactness　of　the　backfill　layer　is　critical　for　ensuring　the　safety　of　the　tunnel　construction.　However,　poor　fluidity　of　cement　generally　leads　to　uneven　grouting　effects,　resulting　in　lower　density　in　certain　areas　of　the　backfill　layer.　In　contrast,　enzyme-induced　carbonate　precipitation　(EICP)　is　an　environmentally　friendly　and　sustainable　technique　which　has　superior　mobility　and　diffusivity　compared　to　cement.　To　investigate　the　reinforcement　effects　of　EICP　technique　on　backfill　layer,　a　series　of　bio-cemented　sand　column　tests　and　model　tests　were　conducted　in　this　study.　The　optimal　working　range　of　pea　gravel　and　sand　for　effective　bio-cementation　were　determined　by　comparing　the　permeability,　unconfined　compressive　strength　(UCS),　calcium　carbonate　content　(CCC),　and　wave　velocity　of　bio-cemented　sand　columns.　The　effects　and　homogeneity　of　reinforcement　based　on　model　tests　were　assessed　by　point　load　tests,　wave　velocity　measurements,　and　calcium　carbonate　content　evaluations.　The　model　tests　with　different　grouting　hole　layout　density　were　conducted　to　obtain　the　optimal　hole　placement　scheme.　The　column　test　results　demonstrated　that　the　optimal　working　range　of　pea　gravel　to　sand　ratio　for　effective　bio-cementation　is　1.25-1.5.　As　the　number　of　grouting　cycles　increases,　the　point　load　strength,　wave　velocity,　CCC　and　UCS　of　the　specimens　increase　while　the　permeability　of　the　specimens　decreases.　The　point　load　strength　of　bio-cemented　specimens　could　reach　up　to　16.53　MPa,　while　the　permeability　was　reduced　by　three　orders　of　magnitude　compared　with　that　of　untreated　specimens.　The　EICP　has　been　demonstrated　to　be　an　effective　technique　capable　of　improving　the　compactness　and　strength　of　the　backfill　layer,　with　aggregates　effectively　cemented　by　calcium　carbonate　generated.　The　model　test　results　demonstrated　that　the　full-coverage　arrangement　hole　scheme　achieves　uniform　cementation,　while　the　space　arrangement　hole　scheme　produces　concentrated　CaCO3　near　grouting　holes.　Furthermore,　a　new　and　improved　grouting　scheme　is　proposed　based　on　the　model　test　results.　The　data　obtained　in　this　study　offer　valuable　references　for　the　reinforcement　of　the　backfill　layer　in　TBM　tunnelling　using　the　EICP　technology.