Previously, electro-conjugate fluid (ECF) micropumps with triangular prism and slit electrode pairs (TPSEs) have been developed and utilized for various mechanical, optical, chemical, and biomedical applications. However, the structural parameters of TPSEs have been determined by trial-and-error rather than systematical analysis. To this end, we select four structural parameters (i.e. hole width of a slit electrode: w, hole depth of a slit electrode: t, tip angle of a triangular prism electrode: , and gap between the triangular prism electrode and slit one: d) and implement the optimization to investigate their effects on the characteristic performance of ECF micropumps mainly depending on the combination of MEMS fabrication and experimental validation. Firstly, as a supplementary means, the simulation analysis is conducted with just consideration of the electric field intensity generated by TPSEs. After MEMS fabrication, the characteristic performance is experimentally characterized by comprehensively considering the generated electric field intensity, the flow dynamics, and the defect of microfabrication. The results show that the designed ECF micropump can attain the maximal output power under an applied DC voltage of 2 kV at the conditions of the slit hole width w = 200 µm, the slit hole depth t = 200 µm, the triangular tip angle = 60°, and the electrode gap d =200 µm. This optimization of TPSEs offers the helpful design guidance of ECF micropumps with higher output performance and promotes their further widespread use.