Prolidase　(PLD)　plays　a　crucial　role　as　a　dipeptidase　in　various　physiological　processes,　specifically　involved　in　the　cleavage　of　proline-containing　dipeptides　for　efficient　recycling　of　proline.　The　accurate　determination　of　PLD　activity　holds　significant　importance　in　clinical　diagnosis.　Herein,　a　solid-state　electrochemiluminescence　(ECL)　biosensor　was　developed　to　address　the　urgent　need　for　PLD　assay.　The　Ru(bpy)32+　was　electrophoretically　deposited　within　the　nanochannels　of　vertically-ordered　mesoporous　silica　film　(VMSF)　on　indium　tin　oxide　(ITO)　electrodes.　The　Ru(bpy)32+-deposited　VMSF/ITO　(Ru-VMSF/ITO)　exhibited　a　remarkable　ECL　response　towards　proline,　attributed　to　the　enhanced　concentration　of　the　reactants　and　improved　electron　transfer　resulting　from　the　nanoconfinement　effect.　As　PLD　specifically　enzymolyzed　the　Gly-Pro　dipeptide　to　release　proline,　a　proline-mediated　biosensor　was　developed　for　PLD　assay.　Increased　PLD　activity　led　to　enhanced　release　of　proline　into　the　porous　solid-state　ECL　sensors,　resulting　in　a　more　robust　ECL　signal.　There　was　a　linear　relationship　between　ΔECL　intensity　and　logarithmic　concentration　of　PLD　in　the　range　of　10–10000　U/L,　with　a　detection　limit　of　1.98　U/L.　Practical　tests　demonstrated　the　reliability　and　convenience　of　the　proposed　bioassay,　making　it　suitable　for　widespread　application　in　PLD　assays.　©　2023　Elsevier　B.V.