Impregnation of a stationary phase by organic and inorganic agents in high-performance thin-layer chromatography (HPTLC) may result in higher separation selectivity and resolution. This work is devoted to the application of novel core–shell polymers consisting of hyperbranched (poly(ethyleneimine)) core surrounded by oligosaccharide shell (PEI-OS) as a component of silica stationary phase in HPTLC for the separation of water-soluble vitamins, amino acids, and chiral β-blocker enantiomers. The influence of polymer structure (degree of substitution of PEI terminal amino groups with oligosaccharides and molecular weight of dendritic core [5 or 25 kDa]) as well as the content of PEI-OS in the stationary phase and a method of modification of the stationary phase on the efficiency of vitamin and amino acid determination and on the enantioselectivity factors of β-blockers separation were investigated. It was established that such polymers can be used as modifying agents of HPTLC systems for on-line preconcentration of vitamins (B2) and amino acids (lysine, tryptophan, and glutamic acids). These polymers can also be recommended as chiral selectors for the effective TLC separation of β-blockers.