The effect of organic solvent on Ru(bpy)i+ chemiluminescent (CL) reactions is investigated in a flow injection system with oxalate and proline as model analytes. The organic solvents chosen for this study are acetonitrile, methanol, 2-propanol and acetone. It is found that all the four organic solvents tested can be used as organic modifiers in HPLC systems, which employ Ru(bpy)i+ chemiluminescence detection. Increasing the concentrations of acetonitrile, methanol and acetone in the phosphate carrier streams from 0 to 30% (v/v) results in increased CL intensities without significant increases in the background signals. In contrast, use of 2-propanol in the carrier streams from 0 to 30% (v/v) results in lower CL intensities, along with dramatic increase in the background signals. Stopped-flow experiments are performed to compare the quantum efficiency of Ru(bpy):+ CL in 100% aqueous phosphate buffer and in 30% acetonitrile modified buffer. Increased quantum efficiency of Ru(bpy)F CL is observed in the acetonitrile modified buffer than that in the 100% aqueous phosphate buffer. Further studies by absorbance and fluorescence experiments reveal that the increased CL intensity in the acetonitrile modified buffer is attributed primarily to the increased excitation quantum efficiency of the Ru(bpy)i+ CL.