Flex-on-board (FOB) interconnection plays an important role in display applications. Due to the thinness of the interconnect, the contact resistance of anisotropic conductive film (ACF) joints is very small; however, the transmission property of ACF joints are still unknown. In this study, we designed a novel coplanar waveguide structure to characterize the transmission properties of ACF joints in FOB interconnections. Specifically, we compared the differences between the Au/Ni coated polymer ball joint and low melting point Sn58Bi solder ball joint in terms of their high frequency performance. Both were bonded under the same conditions of 200 C for 10 s at 2 MPa. A high frequency test was then carried out in the range of 300 MHz to 20 GHz. The results showed that 14 GHz was the resonant frequency of FOB package that contained the Au/Ni coated polymer ball joints, while 1 GHz increased by using Sn58Bi solder joints in FOB package. To our knowledge, there is the first study to determine the high frequency performance of flex-on-board interconnections using ACFs joints and the coplanar waveguide method.