A pump-free and low-cost negative pressure sampling device for injecting well-defined non-biased sample plugs into the separation channel of NICE was developed. It was composed of a pipet bulb, a 3-way electromagnetic valve and a single voltage supply at constant voltage. A sub-atmospheric pressure was created by hand-pressing air out of the pipet bulb and retained in it by switching the 3-way electromagnetic valve at cutoff position. During the sample loading stage, the sub-atmospheric pressure in the pipet bulb was applied via a 3-way electromagnetic valve to the headspace of the sealed sample waste reservoir (SW). A pinched sample plug was formed at the channel intersection in less than 0.5 s. Once the 3-way electromagnetic valve was switched to connect SW to ambient atmosphere to release the vacuum in SW, electrophoresis separation was consequently activated under the electric potentials applied. Experimental results demonstrated the pump-free negative pressure sampling device worked well in a wide vacuum degree ranged from -250 to -30 mbar with a satisfactory analytical precision. The sample consumption for each cycle was calculated to be 51-12 nL under the sampling pressure. Theoretical deduction indicates that the volume of the pipet bulb can be further reduced to 1 mL, which is critical for minimizing the sampling device for MCE.