The popularity of capillary electrochromatography (CEC) has led to an increasing number of studies on the development and evaluation of enantioselective CEC systems. Herein, a novel, simple, and economical method for the preparation of chiral stationary phases for enantioselective open tubular capillary electrochromatography (OTCEC) was reported for the first time. This novel capillary column was fabricated through layer-by-layer self-assembly of GNPs on a 3-mercaptopropyl-trimethoxysilane (MPTMS)-modified fused-silica capillary and subsequent surface functionalization of the GNPs through self-assembly of thiols β-cyclodextrin (SH-β-CD). With this method, monolayer and multilayer GNPs film capillary columns were prepared and evaluated using meptazinol and its three intermediate enantiomers as templates. Consequently, the three-layer GNPs capillary column was found to exhibit favorable enantioseparation performance. Factors that influence the chiral separation resolution were examined. Under the optimized conditions, enantioselectivity was obtained for meptazinol and its three intermediate enantiomers (intermediates Ⅱ, Ⅲ and Ⅳ) in less than 20min with resolutions of 2.05, 0.36, 1.67, 1.53, respectively. The proposed column revealed adequate repeatability concerning run-to-run and day-to-day. In brief, these results confirmed the use of GNPs as the electrochromatographic support can enhance the phase ratio of OTC column in capillary electrochromatography (CEC). Then, this proposed method was well validated with good linearity (≥0.999), recovery (92.0–94.5%) and repeatability, and was successfully used for enantioseparation of meptazinol in spiked urine samples, which indicated the new column's potential usage in biological analysis. Display omitted •Novel β-CD-GNPs modified OTC columns were carried out in CEC.•Monolayer and multilayer GNPs films capillaries were prepared and compared.•The novel OTCEC columns were evaluated by chiral separation of enantiomers.•Key factors affecting the enantioseparation were evaluated and optimized.•The novel OTC capillaries exhibits good reproducibility and excellent stability.