Single cell copy number variations (CNVs), major dynamic changes in humans, result in differential levels of gene expression and account for adaptive traits or underlying disease. Single cell sequencing is needed to reveal these CNVs but has been hindered by single-cell whole-genome amplification (scWGA) bias, leading to inaccurate gene copy number counting. In addition, most of current scWGA methods are labor-intensive, time-consuming and expensive with limited wide application. Here, we report a novel single-cell whole genome library preparation approach based on digital microfluidics for digital counting of single-cell Copy Number Variation (dd-scCNV Seq). dd-scCNV Seq directly fragments the original single-cell DNA and uses these fragments as templates for amplification. These reduplicative fragments can be filtered computationally to generate the original partitioned unique identified fragments, thereby enabling digital counting of copy number variation. dd-scCNV Seq showed an increase in uniformity in the single-molecule data leading to more accurate CNV patterns compared to other methods with low-depth sequencing. Benefiting from digital microfluidics, dd-scCNV Seq allows automated liquid handling, precise single-cell isolation, high-efficiency and low-cost genome library preparation. dd-scCNV Seq will accelerate biological discovery by enabling accurate profiling of copy number variations at single cell resolution.