The trend to integrate more electronic components in a limited space range motivates the development of advanced electronic packaging. Conventional electronic packaging is difficult to perform in stable interfacing interconnection pitch below 7 µm due to the possibility of short‐circuit problems. Herein, a kind of anisotropic conductive film (μ‐ACF) containing periodically arranged conductive microspheres for microscale pitch electrical interfacing interconnection is developed. The periodic arrangement can avert the contact of conductive microspheres, thus enabling stable interconnection with a pitch as small as 5 µm. By coating conductive microspheres with a layer of silicon insulation, the pitch can be further reduced to 3 µm, which can prevent the formation of conductive pathways between electrodes even if they come into contact with each other. Such high‐quality arrangement is achieved by lithography and capillary self‐assembly method, which is expected to be used in high‐throughput production. Thanks to the delicate design, the μ‐ACF can achieve a low contact resistance of 4.62 mΩ mm−2 and a spatial resolution of 3 µm without short‐circuit failure. The spatial resolution can be further improved by adjusting the size of conductive microspheres, which is conducive to the development of highly integrated devices.