In this study, multiple particle binding-liposomes (MPB-Lips), encapsulating the luminophore tris(2',2-bipyridyl)ruthenium (II) complex ([Ru(bpy)]), were developed as an electrochemiluminescence (ECL) signal amplifier and were applied to detect the model analyte streptavidin (SA) using the indirect competitive ECL method. The MPB-Lips were prepared by mixing various ratios of two different liposomes-one containing a phospholipid with a primary amine group and a biotinyl group (BIO/NH-Lip) and one containing a phospholipid with an N-hydroxysuccinimide group (NHS-Lip) to allow binding between particles via amide bonds. Quartz crystal microbalance analysis using SA-modified gold-coated quartz crystals showed that the frequency shift values of MPB-Lips gradually decreased in the order BIO/NH-Lip:NHS-Lip = 1:0 < 1:1 < 1:3 < 1:5. This indicated that MPB-Lips were successfully formed. The indirect competitive ECL method using SA-modified gold electrodes showed that the 1:5-Lip system had greater sensitivity than the 1:0-Lip system-the limit of detection and quantification values for the systems were 1.84 and 6.30 μg mL for 1:0-Lip, and 1.20 and 1.74 μg mL for 1:5-Lip. Finally, the recovery of SA spiked in fetal bovine serum samples using the 1:5-Lip system showed good accuracy and precision with a recovery rate of 83-106% and relative standard deviation of 4-14%. Our study demonstrated that the MPB-Lips system was an effective and useful ECL amplifier and the ECL method using MPB-Lips could be applied to detect an analyte in a real sample.