To improve the concentration sensitivity of cationic analytes in microchip electrophoresis, on-line sample preconcentration by a large-volume sample stacking with an electroosmotic flow pump (LVSEP) was performed using non-aqueous background solutions (BGSs) in cationic polymer-coated microchannels. In the LVSEP analyses using dimethyl sulfoxide-based BGS, however, poly(vinyl alcohol)/poly(allylamine) mixture-coated microchannel gave insufficient enrichments and broader peaks for cationic histamine. By employing a methanolic BGS containing (hydroxypropyl)methyl cellulose, on the other hand, good enrichments of histamine were achieved by LVSEP with the sensitive enhancement factor of 67. To our knowledge, this is first time that LVSEP analyses of cationic analytes were successfully realized in non-aqueous media.