In this work, we chose tris(2,2′-bipyridyl)ruthenium(II)hexafluorophosphate(Ru(bpy)3(PF6)2), a metal-organic complex material,to prepare nanowires, which were subsequently applied for the construction of electrochemiluminescence(ECL) biosensor by immobilizing them onto a glassy carbon electrode(GCE) with graphene-Nafion composite films. The graphene therein, being a two-dimensional carbon nanomaterial with outstanding electronic properties, can obviously improve the conductivity of the Nafion film, as well as enhance the electrochemical signal and ECL intensity of the Ru(bpy)3(PF6)2 nanowires(RuNWs) at low graphene concentration. The developed biosensor exhibited excellent ECL stability with tripropylamine(TPrA) as co-reactant. The ECL biosensor exhibited high sensitive ECL response in a wide linear range and low detection limit for the detection of proline. It is considered that the oxidation products of proline would be responsible for the ECL enhancement. The large electro-active area of the nanowires and the enhancement effect of the graphene played critical roles in the high detection performance of the ECL biosensor. The results demonstrated herein may provide a useful enlightenment for the design of more sensitive ECL biosensors. In this work, we chose tris (2,2’-bipyridyl) ruthenium (II) hexafluorophosphate (Ru (bpy) 3 (PF6) 2), a metal-organic complex material, to prepare nanowires, which were subsequently applied for the construction of electrochemiluminescence (ECL) biosensor by immobilizing them onto a glassy carbon electrode (GCE) with graphene-Nafion composite films. capable of improving two-dimensional carbon nanomaterial with outstanding electronic properties, can obviously improve the conductivity of the Nafion film, The developed biosensor exhibited excellent ECL stability with tripropylamine (TPrA) as co-reactant. The ECL biosensor has high sensitive ECL response in a wide linear range and low detection limit for the detection of proline. It is considered that the oxidation products of proline would be responsible for the ECL enhancement. The large electro-active area of ​​the nanowires and the enhancement effect of the graphene played critical roles in the high detection performance of the ECL biosensor. The results suggests here may provide a useful enlightenment for the design of more sensitive ECL biosensors.