Abstract Motivation The recent introduction of a barcoding protocol for Oxford Nanopore sequencing has increased the versatility of the technology. Several bioinformatics tools have been developed to demultiplex barcoded reads, but none of them supports streaming analysis. This limits the use of multiplexed sequencing in real-time applications, which is one of the main advantages of the technology. Results We introduced npBarcode, an open source and cross-platform tool for barcode demultiplexing in streaming fashion that can be used to pipe data to further real-time analyses. The tool also provides a friendly graphical user interface by integrating the module into npReader, making possible to monitor the progress concurrently when the sequencing is still in progress. We show that our algorithm achieves accuracies at least as good as competing tools. Availability and implementation npBarcode is bundled in Japsa—a Java tools kit for genome analysis, and is freely available at https://github.com/mdcao/japsa. Supplementary information Supplementary data are available at Bioinformatics online.