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In this paper, we propose a method for interconnecting soft polyimide (PI) electrodes using anisotropic conductive films (ACFs). Reliable and automated bonding was achieved through development of a desktop thermocompressive bonding device that could simultaneously deliver appropriate temperatures and pressures to the interconnection area. The bonding conditions were optimized by changing the bonding temperature and bonding pressure. The electrical properties were characterized by measuring the contact resistance of the ACF bonding area, yielding a measure that was used to optimize the applied pressure and temperature. The optimal conditions consisted of applying a pressure of 4 kgf/cm2 and a temperature of 180 °C for 20 s. Although ACF base bonding is widely used in industry (e.g., liquid crystal display manufacturing), this study constitutes the first trial of a biomedical application. We performed a preliminary in vivo biocompatibility investigation of ACF bonded area. Using the optimized temperature and pressure conditions, we interconnected a 40-channel PI multielectrode device for measuring electroencephalography (EEG) signals from the skulls of mice. The electrical properties of electrode were characterized by measuring the impedance. Finally, EEG signals were measured from the mice skulls using the fabricated devices to investigate suitability for application to biomedical devices.