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Single cell analysis (SCA) is nowadays recognized as one of the key tools for diagnostics and fundamental cell biology studies. The Layer-by-layer (LbL) polyelectrolyte assembly is a rather new but powerful technique to produce multilayers. It allows to model the extracellular matrix in terms of its chemical and physical properties. Utilization of the multilayers for SCA may open new avenues in SCA because of the triple role of the multilayer film: (i) high capacity for various biomolecules; (ii) natural mimics of signal molecule diffusion to a cell and (iii) cell patterning opportunities. Besides, light-triggered release from multilayer films offers a way to deliver biomolecules with high spatio-temporal resolution. Here we review recent works showing strong potential to use multilayers for SCA and address accordingly the following issues: biomolecule loading, cell patterning, and light-triggered release.