Considering the advantages and research status of microfluidic chip coupling with mass spectrometry (MS), a microfluidic chip-based multi-channel ionization (MCMCI) for the extraction of untreated compounds in complex matrices without sample pretreatments was developed. Quantitative analysis of human urine spiked with various rhodamine B concentrations was also performed, and good linearity was obtained. Comparing to the macro ionization device, MCMCI significantly improved the integration of ionization source, simplified the operation of such a device, and greatly increased the signal intensity with much lower gas pressure. Comparison of our MCMCI with two and three gas channels indicated that the liquid-liquid extraction process before spraying and after spraying produced similar MS results. Moreover, this MCMCI with three gas channels also implemented simultaneous dual sprays with high DC voltages, the interference of two samples was minor and ion suppression effect was drastically alleviated. Such advantages may easily enable internal calibration for accurate mass measurement. Furthermore, dual extraction can be implemented by integrating such multi-spray configuration, which can improve the extracted signal intensity and sensitivity. These technologies open up new avenues for the application of microfluidic chip coupling with MS.