The qPrimerDB database (real-time quantitative PCR Primer Database, https://biodb.swu.edu.cn/qprimerdb) is the most comprehensive qPCR primer database available to date, with a web front-end providing gene-specific and pre-computed primer pairs across 212 important organisms, including human, mouse, zebrafish, yeast, thale cress, rice, and maize. The qPrimerDB provides an interactive and information-rich web graphical interface to display search and BLAST results as table-based descriptions and associated links. In this database, we provide 4,819,011 of the best primer pairs for each gene based on primer pair coverage (PPC), as well as 68,653,054 alternative gene-specific primer pairs, which can be conveniently batch downloaded. We validated the specificity and efficiency of the qPCR primer pairs for 66 randomly selected genes in six different organisms through qPCR assays and gel electrophoresis. The qPrimerDB database represents a valuable, timesaving resource for gene expression analysis. This resource, which will be updated routinely.