An on-line cyclodextrin assisted sweeping-micellar electrokinetic chromatography (CD assisted sweeping-MEKC) was developed for the simultaneous separation and concentration of four neutral analytes (erianin, dendrophenol, naringenin and scoparone) in Dendrobium officinale Kimura et Migo (D. officinale). The D. officinale was directly determined by this on-line stacking method after simple extraction and dilution. The optimized background solution (BGS) was 50 mM phosphoric acid (PA) containing 100 mM SDS and 30% (v/v) methanol. The best separation and concentration performance of analytes dissolved in 90 mM CD and 100 mM PA was achieved in a short analysis time when injected at 50 mbar for 100 s. Compared with conventional sweeping-MEKC and MEKC method, significant improvement in enrichment efficiency was achieved by using this proposed method. A series of validation studies of the present method was performed under the optimal conditions. Good linearities were obtained with the correlation coefficients in the range of 0.994–0.999, the detection limits were ranged from 13 to 40 ng/mL. Sensitivity enhancement factors (SEFs) were in the range of 28.5–46.8 compared with traditional injection (injection time 3 s). Therefore, the proposed method was successfully applied for the separation and concentration of neutral analytes in real samples. Display omitted •An on-line CD assisted sweeping-MEKC method is developed for separation and concentration of neutral analytes.•Satisfactory resolution and enrichment efficiency are achieved in a short analysis time.•Compared with conventional sweeping-MEKC and MEKC method, the proposed method shows higher enrichment efficiency.