Single-cell RNA sequencing (scRNA-seq) plays a critical role in revealing genetic expression patterns at the single-cell level for cell type identification and rare transcript detection. Although there have been great advances in scRNA-seq methodologies, existing technologies still suffer from complexity and high cost, and an integrated platform for complete library construction is still lacking. Herein we describe Cilo-seq for high-performance scRNA-seq library construction in a single device with programmed and addressable droplet handling based on digital microfluidics. The platform is simulta-neously accessible for convenient single-cell isolation, efficient nucleic acid amplification, low-loss nucleic acid purification and high-quality library preparation by leveraging specific interface design, tiny reaction volume, auxiliary magnetic field control and accurate droplet control. With a closed hydrophobic interface, the platform further reduces nucleic acid loss and exogenous background interference. Cilo-seq provides excellent detection sensitivity (1.4-fold improvement over tube-based methods), accuracy (R=0.98) and cost efficiency (10-fold decrease in cost compared to tube-based methods), and holds great promise for studies of single-cell RNA biology.