The new nano-HPTLC method was proposed for improved separation of patulin from the admixture of the three naturally occurring natural components of fruit juices, which in last years could be often obtained from the internet functional food stores and pharmacies. Separation of patulin (P) from quercetin (Q), ascorbic acid (A) and 5-hydroxymethylfurfural (H) was performed using three different ready-for-use plates NanoAdamant (NA), Nano-SIL-20 (NSIL) and Nano-Durasil-20 (ND) covered with the non-modified silica gel layer. After development on the 8.0 cm distance, the nano-HPTLC plates were sprayed with 0.5% aqueous solution of the 3- methyl-benzothiazolinone hydrazone hydrochloride hydrate (MBTH). Most satisfactory separation of patulin from the each mentioned compounds was obtained using the Nano-SIL-20 plates and toluene-ethyl acetate-98% formic acid (25:20:5, v/v/v) as the mobile phase. In this best nano-HPLTC method the limit of detection of patulin was near 2.5 times lower (4 ng/spot) in comparison to other used here chromatographic systems. Thus, this optimized nano-HPTLC mode could be used for analysis of the solid-phase extracts of patulin from the fresh apples, clarified apple juices and other fruit products.