Single-cell RNA sequencing (scRNA-seq) has become one of the most powerful tools to understand the heterogeneity of biological systems. While barcoding strategies have revolutionized the field of high-throughput scRNA-seq, it is still challenging to achieve highly efficient, direct and universal cell barcoding with cost-effectiveness and minimal sample loss. Herein, a single micro-particle dispenser approach for rapid single barcode bead/cell manipulation and pairing, enabling highly efficient cell barcoding for scRNA-seq (Dispen-Seq) was developed. Notably, Dispen-Seq provides a versatile platform which can enrich cell subgroups of interest while unlimited by input sample amounts, and can respond to changes in sample composition with high resolution and reproducibility. It is anticipated that Dispen-Seq will increase the scope of scRNA-seq from academic research to practical applications.