We present an attolitre-sized arrayed lipid bilayer chamber system (aL-ALBiC) for rapid and massively parallel single-molecule assay of membrane transporter activity. Because of the small reaction volume (200 aL), the aL-ALBiC performed fast detection of single transporter activity, thereby enhancing the sensitivity, throughput, and accuracy of the analysis. Thus, aL-ALBiC broadens the opportunities for single-molecule analysis of various membrane transporters and can be used in pharmaceutical applications such as drug screening.