Microscale biopatterning enables regulation of cell-material interactions and cell shape, and enables multiplexed high-throughput studies in a cell- and reagent-efficient manner. The majority of available techniques rely on physical contact of a stamp, pin, or mask with mainly a dry surface. Inkjet and piezoelectric printing is carried out in a non-contact manner but still requires a substantially dry substrate to ensure fidelity of printed patterns. These existing methods, therefore, are limited for patterning onto delicate surfaces of living cells because physical contact or substantially dry conditions are damaging to them. Microfluidic patterning with laminar streams does enable non-contact patterning in fully aqueous environments but with limited throughput and reagent diffusion across interfacial flows. Here, we describe a polymeric aqueous two-phase system that enables patterning nanolitres of a reagent-containing aqueous phase, in arbitrary shapes, within a second aqueous phase covering a cell monolayer. With the appropriate medium formulation, reagents of interest remain confined to the patterned phase without significant diffusion. The fully aqueous environment ensures high reagent activity and cell viability. The utility of this strategy is demonstrated with patterned delivery of genetic materials to mammalian cells for phenotypic screening of gene expression and gene silencing.