In　this　paper,　we　report　a　layer-by-layer　(LbL)　molecular　imprinting　strategy　for　constructing　molecular　recognition　sites　at　the　surface　of　colloidal　silica　particles　by　glutaraldehyde　(GA)-mediated　covalent　assembly　of　gelatin　protein　in　aqueous　media.　Accompanying　the　repeated　coating　of　gelatin　at　the　amine-capped　silica　particles,　2,4,6-trinitrotoluene　(　TNT)　templates　were　synchronously　imprinted　into　the　formed　gelatin　shells　by　the　charge-transfer　interaction　between　the　electron-deficient　aromatic　rings　of　TNT　and　the　electron-rich　amino　groups　of　gelatin　chains.　The　effective　molecular　recognition　sites　generated　at　the　protein　interlayers　of　gelatin　shells　of　monodisperse　silica@gelatin　particles,　and　the　rebinding　TNT　capacities　changed　nonlinearly　with　the　layer　number　of　imprinted　gelatin.　Three　layers　of　imprinted　gelatin　produced　the　largest　imprinting　factor　of　similar　to　3.0,　which　is　explained　by　the　covalent　assembly　mechanism.　The　imprinting　protocol　is　applicable　to　a　broad　range　of　biomaterials　(　such　as　proteins,　enzymes,　chitosan　and　biopolymers)　for　imprinting　various　molecules　in　aqueous　media.　Therefore,　these　results　reported　here　will　open　a　new　window　of　interest　in　the　exploration　of　novel　molecular　recognition　systems　for　application　in　chemosensors,　selective　separation,　and　drug　screening　and　release.