A liposome immunoblotting assay utilizing an enhanced chemiluminescent reaction was developed by means of substrates permeable through the phospholipid membrane for rapid, automatic and sensitive detection of analytes. In the enhanced chemiluminescent (ECL) reaction, the total amount of chemiluminescent light increased by means of substrate solutions containing several surfactants, such as Tween 20 and TritonX-100. In this liposome immunoblotting assay, clear chemiluminescence signals could be detected from blotted circles on the membrane and increased with the concentration of Tween 20 (0-1%) in the substrate solutions. The sensitivity in the liposome immunoblotting assay was 20 times that of conventional assay using HRP-conjugated antibody, and 2 pg of human IgM as a model analyte could be detected under optimal conditions. Thus, the liposome immunoblotting assay utilizing the ECL reaction has a high sensitivity among other immunoblotting assays and is useful for rapid detection of trace amounts of analytes and low affinity interactions in micro-array and other detection formats.