Increasing research efforts have been recently devoted to the coupling of microfluidic chip-integrated ionization sources to mass spectrometry (MS). Considering the limitations of microfluidic chips coupled with MS such as liquid spreading, dead volume, and manufacturing troubles, this paper proposed a new three-dimensional (3D) flow focusing (FF)-based microfluidic ionizing source. This source was fabricated by using the two-layer soft lithography method with the nozzle placed inside the chip. The proposed FF microfluidic chip can realize two-phase FF with liquid in air regardless of the viscosity ratio of the continuous and dispersed phases. MS results indicated that the proposed FF microfluidic chip can work as a typical electrical ionization source when supplied with high voltage and can serve as a sonic ionization source without high voltage. The electro-sonic FF ionization microfluidic chip is expected to have various applications, particularly in the integrated and portable applications of ionization sources coupling with portable MS in the future.