Single-cell whole-genome sequencing (scWGS) plays a critical role in revealing intercellular genome variations. However, the existing scWGS technologies suffer from tedious single-cell isolation and nonuniform amplification. Here, we present an Integrated single-tube operation for rapid and uniform single-cell whole-genome sequencing (Iso-seq) to overcome these limitations. The specially designed dual-well structure in the cap is based on size exclusion and locally quasi-hydrodynamic principles, which ensure that one cell is trapped in one cap, while the capillary-based centrifugal-driven droplet generator facilitates uniform droplet generation in the tube. Using Iso-seq, single cells can be isolated by the dual-well structure in an eight-cap strip within 3 min and the corresponding whole-genome of single cells can be readily compartmentalized in parallel droplets. The prepared single-cell samples show a high mapping ratio, broad coverage of the genome, and low coefficient of variation, demonstrating the good performance of Iso-seq in scWGS. With the advantages of rapid single-cell isolation, convenient droplet generation, broad coverage of the genome, and excellent sequencing uniformity, Iso-seq is promising for a wide variety of biological applications.