Three-dimensional porous graphene (3D-pGR) was used to immobilize Ru(II) tris-bipyridyl [Ru(bpy) 3 2+ ] for electrochemiluminescence (ECL) based sensing of tripropylamine (TPA). The 3D-pGR with interpenetrating porous structures was produced from freeze-drying dispersions containing graphene oxide followed by calcination. Field emission scanning electron microscopy, Raman spectroscopy and X-ray photoelectron spectroscopy were used to characterize morphologies and the composition of the 3D-pGR. After immobilization of Ru(bpy) 3 2+ onto the electrodes, the results indicated that the ECL intensity from the 3D-pGR modified electrode was higher by a factor of 2.5 than that from the graphene film, which was attributed to a larger active surface area and faster electron transfer. The modified electrode was further used to determine TPA in water, and a good linear range from 0.5 to 100 μM with the detection limit as low as 0.4 nM was obtained. The ECL assay presented here also exhibits potential applications in detection of biomolecules such as DNA, glucose, thrombin, lysozyme, etc.