Microflow visualization (μ-FV) has been performed to study the silver nanoparticle droplet ejected from a drop-on-demand piezoelectric inkjet printhead and the equilibrium line characteristic of the nano-silver droplet deposition on PI substrates. The unipolar waveform with a frequency of 1000 Hz and an amplitude of 60 V has been adopted for ejecting the silver nanoparticle droplet with a solid content of 30%, surface tension of 30 mN/m and viscosity of 15 cps. The back pressure is modulated to prevent the formation of satellite droplets experimentally. The PI substrate was placed onto a computer-controlled three-axis moving stage capable of a movement accuracy of 30 μm. Therefore multiple prints of the nano-silver conductive lines have also been carried out based on the moving accuracy. The deposited silver nanoparticle conductive lines with the inter-dot spacings from 25 μm to 45 μm with single print to prints quadruple prints have also been investigated. Besides, the O₂ plasma treatment has been applied on the PI substrates with two durations. After the thermal treatment (sinter temperature of 200°C and sinter duration of 1 hour), the optical microscopic images of the deposited silver nanoparticle conductive lines before and after O₂ plasma treatment have been obtained. For the first time, the quadruple prints of the silver nanoparticle conductive lines on the PI films have been investigated to observe the line widths and electrical resistances. μ-FV has been successfully carried out to study the silver nanoparticle droplets ejected from a piezoelectric ink-jet printhead and the equilibrium film characteristic on PI substratse. The nano-silver conductive lines with quadruple prints on the PI substrate have a line width of 800 μm and a resistance of 1.4 Ω/cm which is also measured on the PI substrate with 2 min O₂ plasma treatment and single print (line width = 680 μm).